# Patent

## Title
Activation of bioluminescence by structural complementation

## Abstract

Provided herein are compositions and methods for the assembly of a bioluminescent complex from two or more non-luminescent (e.g., substantially non-luminescent) peptide and/or polypeptide units. In particular, bioluminescent activity is conferred upon a non-luminescent polypeptide via structural complementation with another, complementary non-luminescent peptide.

## Background
CROSS-REFERENCE TO RELATED APPLICATION
The present application is a continuation of U.S. patent application Ser. No. 14/209,610, filed Mar. 13, 2014, which claims priority to U.S. Provisional Patent Application Ser. No. 61/791,549 filed Mar. 15, 2013, each of which is hereby incorporated by reference in its entirety.


FIELD
Provided herein are compositions and methods for the assembly of a bioluminescent complex from two or more non-luminescent (e.g., substantially non-luminescent) peptide and/or polypeptide units. In particular, bioluminescent activity is conferred upon a non-luminescent polypeptide via structural complementation with another, complementary non-luminescent peptide.
BACKGROUND
Biological processes rely on covalent and non-covalent interactions between molecules, macromolecules and molecular complexes. In order to understand such processes, and to develop techniques and compounds to manipulate them for research, clinical and other practical applications, it is necessary to have tools available to detect and monitor these interactions. The study of these interactions, particularly under physiological conditions (e.g. at normal expression levels for monitoring protein interactions), requires high sensitivity.
SUMMARY
The present invention relates to compositions comprising complementary non-luminescent amino acid chains (e.g., substantially non-luminescent peptides and/or polypeptides that are not fragments of a preexisting protein), complexes thereof, and methods of generating an optically detectable bioluminescent signal upon association of the non-luminescent amino acid chains (e.g., peptides and/or polypeptides). In some embodiments, the present invention provides two or more non-luminescent, or substantially non-luminescent peptides and/or polypeptides, that, when brought together, assemble into a bioluminescent complex. In some embodiments, a pair of substantially non-luminescent peptide and/or polypeptide units assembles into a bioluminescent complex. In other embodiments, three or more substantially non-luminescent peptide and/or polypeptide units assemble into a bioluminescent complex (e.g., ternary complex, tertiary complex, etc.). Provided herein are technologies for detecting interactions between molecular entities (e.g., proteins, nucleic acids, carbohydrates, small molecules (e.g., small molecule libraries)) by correlating such interactions to the formation of a bioluminescent complex of otherwise non-luminescent (e.g., substantially non-luminescent) amino acid chains.
In some embodiments, the assembled pair catalyzes a chemical reaction of an appropriate substrate into a high energy state, and light is emitted. In some embodiments, a bioluminescent complex exhibits luminescence in the presence of substrate (e.g., coelenterazine, furimazine, etc.).
Although the embodiments described herein primarily describe and refer to complementary, non-luminescent amino acid chains that form bioluminescent complexes, it is noted that the present technology can equally be applied to other detectable attributes (e.g., other enzymatic activities, generation of a fluorophore, generation of a chromophore, etc.). The embodiments described herein relating to luminescence should be viewed as applying to complementary, substantially non-enzymatically active amino acid chains (e.g., peptides and/or polypeptides that are not fragments of a preexisting protein) that separately lack a specified detectable activity (e.g., enzymatic activity) or substantially non-enzymatically active subunits of a polypeptide, complexes thereof, and methods of generating the detectable activity (e.g., an enzymatic activity) upon association of the complementary, substantially non-enzymatically active amino acid chains (e.g., peptides and/or polypeptides). Further, embodiments described herein that refer to non-luminescent peptides and/or polypeptides are applied, in some embodiments, to substantially non-luminescent peptides and/or polypeptides.
The invention is further directed to assays for the detection of molecular interactions between molecules of interest by linking the interaction of a pair of non-luminescent peptides/polypeptides to the interaction molecules of interest (e.g., transient association, stable association, complex formation, etc.). In such embodiments, a pair of a non-luminescent elements are tethered (e.g., fused) to molecules of interest and assembly of the bioluminescent complex is operated by the molecular interaction of the molecules of interest. If the molecules of interest engage in a sufficiently stable interaction, the bioluminescent complex forms, and a bioluminescent signal is generated. If the molecules of interest fail to engage in a sufficiently stable interaction, the bioluminescent complex will not form or only form weakly, and a bioluminescent signal is not detectable or is substantially reduced (e.g., substantially undetectable, essentially not detectable, etc.). In some embodiments, the magnitude of the detectable bioluminescent signal is proportional (e.g., directly proportional) to the amount, strength, favorability, and/or stability of the molecular interactions between the molecules of interest.
In some embodiments, the present invention provides peptides comprising an amino acid sequence having less than 100% (e.g., 20% . . . 30% . . . 40% . . . 50% . . . 60% . . . 70% . . . 80%, or more) sequence identity with SEQ ID NO: 2, wherein a detectable bioluminescent signal is produced when the peptide contacts a polypeptide consisting of SEQ ID NO: 440. In some embodiments, the present invention provides peptides comprising an amino acid sequence having less than 100% and greater than 40% (e.g., >40%, >45%, >50%, >55%, >60%, >65%, >70%, >75%, >80%, >85%, >90%, >95%, >98%, >99%) sequence identity with SEQ ID NO: 2, wherein a detectable bioluminescent signal is produced when the peptide contacts a polypeptide consisting of SEQ ID NO: 440. In some embodiments, a detectable bioluminescent signal is produced when the peptide contacts a polypeptide having less than 100% and greater than 40% (e.g., >40%, >45%, >50%, >55%, >60%, >65%, >70%, >75%, >80%, >85%, >90%, >95%, >98%, >99%) sequence identity with SEQ ID NO: 440. In certain embodiments, the detectable bioluminescent signal is produced, or is substantially increased, when the peptide associates with the polypeptide comprising or consisting of SEQ ID NO: 440, or a portion thereof. In preferred embodiments, the peptide exhibits alteration (e.g., enhancement) of one or more traits compared to a peptide of SEQ ID NO: 2, wherein the traits are selected from: affinity for the polypeptide consisting of SEQ ID NO: 440, expression, intracellular solubility, intracellular stability and bioluminescent activity when combined with the polypeptide consisting of SEQ ID NO: 440. Although not limited to these sequences, the peptide amino acid sequence may be selected from amino acid sequences of SEQ ID NOS: 3-438 and 2162-2365. In some embodiments, fusion polypeptides are provided that comprise: (a) an above described peptide, and (b) a first interaction polypeptide that forms a complex with a second interaction polypeptide upon contact of the first interaction polypeptide and the second interaction polypeptide. In certain embodiments, bioluminescent complexes are provided that comprise: (a) a first fusion polypeptide described above and (b) a second fusion polypeptide comprising: (i) the second interaction polypeptide and (ii) a complement polypeptide that emits a detectable bioluminescent signal when associated with the peptide comprising an amino acid sequence having less than 100% and greater than 40% sequence identity with SEQ ID NO: 2; wherein the first fusion polypeptide and second fusion polypeptide are associated; and wherein the peptide comprising an amino acid sequence having less than 100% and greater than 40% sequence identity with SEQ ID NO: 2 and the complement polypeptide are associated.
In some embodiments, the present invention provides polypeptides comprising an amino acid sequence having less than 100% sequence identity with SEQ ID NO: 440, wherein a detectable bioluminescent signal is produced when the polypeptide contacts a peptide consisting of SEQ ID NO: 2. In some embodiments, the present invention provides polypeptides comprising an amino acid sequence having less than 100% and greater than 40% (e.g., >40%, >45%, >50%, >55%, >60%, >65%, >70%, >75%, >80%, >85%, >90%, >95%, >98%, >99%) sequence identity with SEQ ID NO: 440, wherein a detectable bioluminescent signal is produced when the polypeptide contacts a peptide consisting of SEQ ID NO: 2. In some embodiments, a detectable bioluminescent signal is produced when the polypeptide contacts a peptide having less than 100% and greater than 40% (e.g., >40%, >45%, >50%, >55%, >60%, >65%, >70%, >75%, >80%, >85%, >90%, >95%, >98%, >99%) sequence identity with SEQ ID NO: 2. In some embodiments, the polypeptide exhibits alteration (e.g., enhancement) of one or more traits compared to a peptide of SEQ ID NO: 440, wherein the traits are selected from: affinity for the peptide consisting of SEQ ID NO: 2, expression, intracellular solubility, intracellular stability, and bioluminescent activity when combined with the peptide consisting of SEQ ID NO: 2. Although not limited to such sequences, the polypeptide amino acid sequence may be selected from one of the amino acid sequences of SEQ ID NOS: 441-2156. In some embodiments, the detectable bioluminescent signal is produced when the polypeptide associates with the peptide consisting of SEQ ID NO: 2. In some embodiments, a fusion polypeptide is provided that comprises: (a) a polypeptide described above and (b) a first interaction polypeptide that forms a complex with a second interaction polypeptide upon contact of the first interaction polypeptide and the second interaction polypeptide. In certain embodiments, a bioluminescent complex is provided that comprises: (a) a first fusion polypeptide described above; and (b) a second fusion polypeptide comprising: (i) the second interaction polypeptide and (ii) a complement peptide that causes the polypeptide comprising an amino acid sequence having less than 100% and greater than 40% (e.g., >40%, >45%, >50%, >55%, >60%, >65%, >70%, >75%, >80%, >85%, >90%, >95%, >98%, >99%) sequence identity with SEQ ID NO: 440 to emit a detectable bioluminescent signal when an association is formed between the two; wherein the first fusion polypeptide and second fusion polypeptide are associated; and wherein the polypeptide comprising an amino acid sequence having less than 100% and greater than 40% (e.g., >40%, >45%, >50%, >55%, >60%, >65%, >70%, >75%, >80%, >85%, >90%, >95%, >98%, >99%) sequence identity with SEQ ID NO: 440 and the complement peptide are associated.
In some embodiments, the present invention provides nucleic acids (e.g., DNA, RNA, etc.), oligonucleotides, vectors, etc., that code for any of the peptides, polypeptides, fusion proteins, etc., described herein. In some embodiments, a nucleic acid comprising or consisting of one of the nucleic acid sequences of SEQ ID NOS: 3-438 and 2162-2365 (coding for non-luminescent peptides) and/or SEQ ID NOS 441-2156 (coding for non-luminescent polypeptides) are provided. In some embodiments, other nucleic acid sequences coding for amino acid sequences of SEQ ID NOS: 3-438 and 2162-2365 and/or SEQ ID NOS 441-2156 are provided.
In certain embodiments, the present invention provides bioluminescent complexes comprising: (a) a peptide comprising a peptide amino acid sequence having less than 100% sequence identity (e.g., >99%, <95%, <90%, <80%, <70%, <60%, <50%, etc.) with SEQ ID NO: 2; and (b) a polypeptide comprising a polypeptide amino acid sequence having less than 100% and greater than 40% (e.g., >40%, >45%, >50%, >55%, >60%, >65%, >70%, >75%, >80%, >85%, >90%, >95%, >98%, >99%) sequence identity with SEQ ID NO: 440, wherein the bioluminescent complex exhibits detectable luminescence. In certain embodiments, the present invention provides bioluminescent complexes comprising: (a) a peptide comprising a peptide amino acid sequence having less than 100% and greater than 40% (e.g., >40%, >45%, >50%, >55%, >60%, >65%, >70%, >75%, >80%, >85%, >90%, >95%, >98%, >99%) sequence identity with SEQ ID NO: 2; and (b) a polypeptide comprising a polypeptide amino acid sequence having less than 100% and greater than 40% (e.g., >40%, >45%, >50%, >55%, >60%, >65%, >70%, >75%, >80%, >85%, >90%, >95%, >98%, >99%) sequence identity with SEQ ID NO: 440, wherein the bioluminescent complex exhibits detectable luminescence. Although not limited to particular sequences, in some embodiments, the peptide amino acid sequence is selected from one of the amino acid sequences provided in SEQ ID NOS: 3-438 and 2162-2365.
In various embodiments, bioluminescent complexes are provided that comprise: (a) a first amino acid sequence that is not a fragment of a preexisting protein; and (b) a second amino acid sequence that is not a fragment of a preexisting protein, wherein the bioluminescent complex exhibits detectable luminescence, wherein the first amino acid sequence and the second amino acid sequence are associated. Some such bioluminescent complexes further comprise: (c) a third amino acid sequence comprising a first member of an interaction pair, wherein the third amino acid sequence is covalently attached to the first amino acid sequence; and (d) a fourth amino acid sequence comprising a second member of an interaction pair, wherein the fourth amino acid sequence is covalently attached to the second amino acid sequence. In certain embodiments, interactions (e.g., non-covalent interactions (e.g., hydrogen bonds, ionic bonds, van der Waals forces, hydrophobic interactions, etc.) covalent interactions (e.g., disulfide bonds), etc.) between the first amino acid sequence and the second amino acid sequence do not significantly associate the first amino acid sequence and the second amino acid sequence in the absence of the interactions between the first member and the second member of the interaction pair. In some embodiments, a first polypeptide chain comprises the first amino acid sequence and the third amino acid sequence, and wherein a second polypeptide chain comprises the second amino acid sequence and the fourth amino acid sequence. In some embodiments, the first polypeptide chain and the second polypeptide chain are expressed within a cell.
In some embodiments, the present invention provides a bioluminescent complex comprising: (a) a pair of non-luminescent elements, wherein each non-luminescent element is not a fragment of a preexisting protein; (b) an interaction pair, wherein each interaction element of the interaction pair is covalently attached to one of the non-luminescent elements.
Various embodiments described herein provide methods of detecting an interaction between a first amino acid sequence and a second amino acid sequence comprising, for example, the steps of: (a) attaching the first amino acid sequence to a third amino acid sequence and attaching the second amino acid sequence to a fourth amino acid sequence, wherein the third and fourth amino acid sequences are not fragments of a preexisting protein, wherein a complex of the third and fourth amino acid sequences emits a detectable bioluminescent signal (e.g., substantially increased bioluminescence relative to the polypeptide chains separately), wherein the interactions (e.g., non-covalent) between the third and fourth amino acid sequences are insufficient to form, or only weakly form, a complex of the third and fourth amino acid sequences in the absence of additional stabilizing and/or aggregating conditions, and wherein a interaction between the first amino acid sequence and the second amino acid sequence provides the additional stabilizing and/or aggregating forces to produce a complex of the third and fourth amino acid sequences; (b) placing the first, second, third, and fourth amino acid sequences of step (a) in conditions to allow for interactions between the first amino acid sequence and the second amino acid sequence to occur; and (c) detecting the bioluminescent signal emitted by the complex of the third and fourth amino acid sequences, wherein detection of the bioluminescent signal indicates an interaction between the first amino acid sequence and the second amino acid sequence. In some embodiments, attaching the first amino acid sequence to the third amino acid sequence and the second amino acid sequence to the fourth amino acid sequence comprises forming a first fusion protein comprising the first amino acid sequence and the third amino acid sequence and forming a second fusion protein comprising the second amino acid sequence and the fourth amino acid sequence. In some embodiments, the first fusion protein and the second fusion protein further comprise linkers between said first and third amino acid sequences and said second and fourth amino acid sequences, respectively. In certain embodiments, the first fusion protein is expressed from a first nucleic acid sequence coding for the first and third amino acid sequences, and the second fusion protein is expressed from a second nucleic acid sequence coding for the second and fourth amino acid sequences. In some embodiments, a single vector comprises the first nucleic acid sequence and the second nucleic acid sequence. In other embodiments, the first nucleic acid sequence and the second nucleic acid sequence are on separate vectors. In certain embodiments, the steps of (a) “attaching” and (b) “placing” comprise expressing the first and second fusion proteins within a cell.
Provided herein are methods of creating, producing, generating, and/or optimizing a pair of non-luminescent elements comprising: (a) aligning the sequences of three or more related proteins; (b) determining a consensus sequence for the related proteins; (c) providing first and second fragments of a protein related to three or more proteins (or providing first and second fragments of one of the three or more proteins), wherein the fragments are individually substantially non-luminescent but exhibit luminescence upon interaction of the fragments; (d) mutating the first and second fragments at one or more positions each, wherein the mutations alter the sequences of the fragments to be more similar to a corresponding portion of the consensus sequence (e.g., wherein the mutating results in a pair of non-luminescent elements that are not fragments of a preexisting protein), and (e) testing the pair of non-luminescent elements for the absence (e.g., essential absence, substantial absence, etc.) of luminescence when unassociated, and luminescence upon association of the non-luminescent pair into a bioluminescent complex. Examples of such a process are described in Examples 1-5. In some embodiments, the non-luminescent elements exhibit enhancement of one or more traits compared to the first and second fragments, wherein the traits are selected from: increased reconstitution affinity, decreased reconstitution affinity, enhanced expression, increased intracellular solubility, increased intracellular stability, and increased intensity of reconstituted luminescence.
In some embodiments, the present invention provides detection reagents comprising: (a) a polypeptide comprising an amino acid sequence having less than 100% and greater than 40% sequence identity with SEQ ID NO: 440, wherein a detectable bioluminescent signal is produced when the polypeptide contacts a peptide consisting of SEQ ID NO: 2, and (b) a substrate for a bioluminescent complex produced by the polypeptide and a peptide consisting of SEQ ID NO: 2. In some embodiments, the present invention provides detection reagents comprising: (a) a peptide comprising an amino acid sequence having less than 100% sequence identity with SEQ ID NO: 2, wherein a detectable bioluminescent signal is produced when the peptide contacts a polypeptide consisting of SEQ ID NO: 440, and (b) a substrate for a bioluminescent complex produced by the peptide and a polypeptide consisting of SEQ ID NO: 440. In some embodiments, the present invention provides detection reagents comprising: (a) a peptide comprising an amino acid sequence having less than 100% and greater than 40% sequence identity with SEQ ID NO: 2, wherein a detectable bioluminescent signal is produced when the peptide contacts a polypeptide consisting of SEQ ID NO: 440, and (b) a substrate for a bioluminescent complex produced by the peptide and a polypeptide consisting of SEQ ID NO: 440.



BRIEF DESCRIPTION OF THE DRAWINGS
FIG. 1 shows a graph depicting the effect of various mutations of the GVTGWRLCKRISA (SEQ ID NO: 236) peptide on luminescence resulting from complementation with SEQ ID NO: 440.
FIG. 2 shows a graph depicting the effect of various mutations of the SEQ ID NO: 440 polypeptide on luminescence resulting from complementation with GVTGWRLCKRISA (SEQ ID NO: 236) or GVTGWRLFKRISA (SEQ ID NO: 108) peptides.
FIG. 3 (top) shows the luminescence (RLUs) detected in each non-luminescent polypeptide (NLpoly) mutant containing a single glycine to alanine substitution and (bottom) shows the fold increase in luminescence over wild-type.
FIG. 4 (top) show the luminescence (RLUs) detected in each NLpoly mutant containing a composite of glycine to alanine substitutions and (bottom) shows the fold increase in luminescence over wild-type.
FIG. 5 shows a graph depicting the luminescence (RLUs) detected in HT-NLpeptide fusions.
FIG. 6 shows a graph depicting the luminescence (RLUs) detected in HT-NLpep fusions.
FIG. 7 shows a graph depicting the luminescence (RLUs) detected in NLpeptide-HT fusions.
FIG. 8 shows the luminescence (RLUs) generated by a luminescent complex after freeze-thaw cycles of non-luminescent peptide (NLpep).
FIG. 9 shows concentration normalized activity of peptides, and the TMR gel used to determine the relative concentrations.
FIG. 10 shows a graph of the luminescence of various mutations of residue R11 of NLpoly-5A2 in the presence of NLpep53 (top) and in the absence of complimentary peptide (bottom).
FIG. 11 shows a graph of the luminescence of various mutations of residue A15 of NLpoly 5A2 in the presence of NLpep53 (top) and in the absence of complimentary peptide (bottom).
FIG. 12 shows a graph of the luminescence of various mutations of residue L18 of NLpoly 5A2 in the presence of NLpep53 (top) and in the absence of complimentary peptide (bottom).
FIG. 13 shows a graph of the luminescence of various mutations of residue F31 of NLpoly 5A2 in the presence of NLpep53 (top) and in the absence of complimentary peptide (bottom).
FIG. 14 shows a graph of the luminescence of various mutations of residue V58 of NLpoly 5A2 in the presence of NLpep53 (top) and in the absence of complimentary peptide (bottom).
FIG. 15 shows a graph of the luminescence of various mutations of residue A67 of NLpoly 5A2 in the presence of NLpep53 (top) and in the absence of complimentary peptide (bottom).
FIG. 16 shows a graph of the luminescence of various mutations of residue M106 of NLpoly 5A2 in the presence of NLpep53 (top) and in the absence of complimentary peptide (bottom).
FIG. 17 shows a graph of the luminescence of various mutations of residue L149 of NLpoly 5A2 in the presence of NLpep53 (top) and in the absence of complimentary peptide (bottom).
FIG. 18 shows a graph of the luminescence of various mutations of residue V157 of NLpoly 5A2 in the presence of NLpep53 (top) and in the absence of complimentary peptide (bottom).
FIG. 19 shows a graph of the luminescence of NLpep-HT fusions.
FIG. 20 shows a graph of the luminescence of NLpep-HT fusions, and a TMR gel indicating their relative expression levels.
FIG. 21 shows a graph of the luminescence of NLpep-HT fusions.
FIG. 22 shows a graph of the luminescence of NLpoly 5A2 (top) and NLpoly5A2+R11E in the presence of various NLpeps (bottom).
FIG. 23 shows a graph of the luminescence of NLpep-HT fusions.
FIG. 24 shows a graph of the luminescence of NLpolys 1-13 with NLpep53 (top) and without complimentary peptide (bottom).
FIG. 25 shows a graph of the luminescence of various NLpolys with NLpep53 with NANOGLO or DMEM buffer and furimazine or coelenterazine substrate.
FIG. 26 shows a graph comparing luminescence in the presence of a ratio of furimazine with coelenterazine for various NLpolys and NLpep53.
FIG. 27 shows a graph comparing luminescence in the presence of a ratio of furimazine to coelenterazine for various NLpolys and NLpep53.
FIG. 28 shows a graph comparing luminescence in the presence of furimazine with coelenterazine for various NLpolys and NLpep53 in HEK293 cell lysate.
FIG. 29 shows a graph of the luminescence of various combinations of NLpoly and NLpep pairs in DMEM buffer with furimazine.
FIG. 30 shows a graph of the signal/background luminescence of various combinations of NLpoly and NLpep pairs in DMEM buffer with furimazine.
FIG. 31 shows a graph of luminescence and substrate specificity of various NLpoly mutants with NLpep69 using either furimazine or coelenterazine as a substrate.
FIG. 32 shows a comparison of luminescence and substrate specificity of various NLpoly mutants with NLpep69 using either furimazine or coelenterazine as a substrate, and under either lytic (bottom graph) or live cell (top graph) conditions.
FIG. 33 shows a comparison of luminescence and substrate specificity of NLpoly mutants with NLpep78 using either furimazine or coelenterazine as a substrate, and under either lytic (bottom graph) or live cell (top graph) conditions.
FIG. 34 shows a comparison of luminescence and substrate specificity of various NLpoly mutants with NLpep79 using either furimazine or coelenterazine as a substrate, and under either lytic (bottom graph) or live cell (top graph) conditions.
FIG. 35 shows graphs of the luminescence of NLpep78-HT (top) and NLpep79-HT (bottom) fusions in the presence of various NLpolys.
FIG. 36 shows a graph of the luminescence of various NLpolys in the absence of NLpep.
FIG. 37 shows graphs of the luminescence of NLpep78-HT (top) and NLpep79-HT (bottom) fusions in the presence of various NLpolys with either furimazine or coelenterazine substrates.
FIG. 38 shows a graph of the luminescence of NLpep78-HT with various NLpolys expressed in CHO and HeLa cells.
FIG. 39 shows graphs of raw and normalized luminescence from NLpoly fused to firefly luciferase expressed in HEK293, Hela, and CHO cell lysates.
FIG. 40 shows graphs of raw and normalized luminescence from NLpoly fused to click beetle red luciferase expressed in HEK293, Hela, and CHO cell lysates.
FIG. 41 shows a graphs of luminescence of complementation in live cells using either NLpoly wild-type or 5P.
FIG. 42 shows graphs of luminescence of cell-free complementation of NLpep78-HT fusion (top) and NLpep79-HT fusion (bottom) with various NLpolys.
FIG. 43 shows a graph of binding affinities for various combinations of NLpeps and NLpolys expressed in HeLa, HEK293 and CHO cell lysate.
FIG. 44 shows a graph of binding affinities for various combinations of NLpeps and NLpolys in PBS or NANOGLO buffer.
FIG. 45 shows a graph of binding affinities for NLpoly 5P with NLpep9 (SEQ ID NO: 236) or NLpep53 (SEQ ID NO: 324) expressed in HeLa, HEK293 or CHO cell lysate.
FIG. 46 shows a graph of luminescence of varying amounts of NLpolys in the absence of NLpep.
FIG. 47 shows a graph of background luminescence of various NLpoly variants.
FIG. 48 shows a graph of background luminescence of various NLpoly variants.
FIG. 49 shows a SDS-PAGE gel of total lysate and soluble fraction of several NLpoly variants
FIG. 50 shows (a) a SDS-PAGE gel of the total lysate and soluble fraction of NLpoly variants and (b) background luminescence of NLpoly variants.
FIG. 51 shows graphs of the luminescence generated with several NLpoly variants when complemented with 10 nm (right) or 100 nM (left) of NLpep78.
FIG. 52 shows graphs depicting background luminescence in E. coli lysate of various NLpoly variants.
FIG. 53 shows graphs depicting luminescence in E. coli lysate of various NLpoly variants complemented with NLpep78.
FIG. 54 shows graphs depicting luminescence in E. coli lysate of various NLpoly variants complemented with NLpep79.
FIG. 55 shows a graph of signal to background of various NLPolys variants complemented with NLpep78 or NLpep79 and normalized to NLpoly 5P.
FIG. 56 shows a graph depicting background, luminescence with NLpep79 (right) or NLpep78 (left) and signal-to-noise or various NLpoly variants.
FIG. 57 shows a SDS-PAGE gel of the total lysate and soluble fraction in various NLpoly 5P variants.
FIG. 58 shows (A) the amount of total lysate and soluble fraction of NLpoly 5P and NLpoly I107L, (B) luminescence generated by NLpoly 5P or NLpoly I107L without NLpep or with NLpep78 or NLpep79 and (C) the improved signal-to-background of NLpoly I107L over NLpoly 5P.
FIG. 59 shows graphs of luminescence for various NLpoly variants (A) without complementary peptide, (B) with NLpep78-HT and (C) with NLpep79-HT.
FIG. 60 shows graphs of luminescence for various NLpoly variants (A) without complementary peptide, (B) with NLpep78-HT and (C) with NLpep79-HT.
FIG. 61 shows graphs of luminescence for various NLpoly variants (A) without complementary peptide, (B) with NLpep78-HT and (C) with NLpep79-HT.
FIG. 62 shows graphs of luminescence for various NLpoly variants (A) without complementary peptide, (B) with NLpep78-HT and (C) with NLpep79-HT.
FIG. 63 shows binding affinity between an elongated NLpoly variant (additional amino acids at the C-terminus) and a shortened NLpep (deleted amino acids at the N-terminus).
FIG. 64 shows a graph of binding affinity of various NLpoly variants with NLpep78.
FIG. 65 shows the binding and Vmax of NLpep80 and NLpep87 to 5P expressed in mammalian cells (CHO, HEK293T and HeLa).
FIG. 66 shows the binding and Vmax of NLpep80 and NLpep87 to NLpoly 5P expressed in E. coli. 
FIG. 67 shows a graph of luminescence of shortened NLpolys with elongated NLpeps.
FIG. 68 shows graphs of Kd and Vmax of NLpoly 5P in HeLa lysate with various complementary NLpeps.
FIG. 69 shows a graph of binding affinities for several NLpoly variants with NLpep81.
FIG. 70 shows a graph of binding affinities for several NLpoly variants with NLpep82.
FIG. 71 shows a graph of binding affinities for several NLpoly mutants with NLpep78.
FIG. 72 shows a graph of Michaelis constants for several NLpoly mutants with NLpep78.
FIG. 73 shows graphs of luminescence from a tertiary complementation of two NLpeps and NLpoly 5P-B9.
FIG. 74 shows a graph of luminescence of titration of NLpoly 5P with NLpep88-HT.
FIG. 75 shows images of intracellular localization of various NLpep fusions with HaloTag (HT).
FIG. 76 shows images of intracellular localization of NLpoly(wt) and NLpoly(5P).
FIG. 77 demonstrates the ability to detect via complementation an NLPep-conjugated protein of interest following separation by SDS-PAGE and transfer to a PVDF membrane.
FIG. 78 shows a graph of relative luminescent signal from various NLpoly variants compared to NLpoly 5P (in the absence of NLpep).
FIG. 79 shows a graph of relative luminescent signal over background from various NLpolys compared to NLpoly 5P (in the absence of NLpep).
FIG. 80 compares the dissociation constants for NLpeps consisting of either 1 (SEQ ID NO: 156) or 2 (SEQ ID NO: 2580) repeat units of NLpep78.
FIG. 81 shows the affinity between NLpoly 5A2 and NLpep86.
FIG. 82 shows graphs of the luminescence from NLpoly variants without NLpep, with NLpep78, and NLpep79.
FIG. 83-90 show the dissociation constants as well as the Vmax values for NLpoly 5A2, 5P, 8S and 11S with 96 variants of NLpeps. The upper graphs show the values for SEQ ID Nos: 2366-2413. The lower graphs show the values for SEQ ID NOs: 2414-2461.
FIG. 91 shows an image of a protein gel of total lysates and the soluble fraction of the same lysate for NLpoly variants.
FIG. 92 shows an image of a protein gel of total lysates and the soluble fraction of the same lysate for NLpoly variants as well as a table containing the dissociation constants for the same variants.
FIG. 93 shows the substrate specificity for NLpoly 5P and 11S with NLpep79 and demonstrates that NLpoly 11S has superior specificity for furimazine than 5P.
FIG. 94 shows an image of a protein gel that follows the affinity purification of NLpoly 8S through binding NLpep78.
FIG. 95 contains a table of the association and dissociation rate constants for the binding of NLpoly WT or NLpoly 11S to NLpepWT, 78 or 79.
FIG. 96 shows the Km values for various pairs of NLpoly/NLpep.
FIG. 97 compares the dissociation constant for NLpoly 11S/NLpep79 at sub-saturating and saturating concentrations of furimazine.
FIG. 98 compares the Km values for NLpoly 5A2 with NLpepWT, 78 and 79.
FIG. 99 shows the luminescence of NLpolys from various steps in the evolution process in the absence of NLpep.
FIG. 100 shows the improvement in luminescence from E. coli-derived NLpoly over the course of the evolution process with an overall  ̃105 improvement (from NLpolyWT:NLpepWT to NLpoly11S:NLpep80).
FIG. 101 shows the improvement in luminescence from HeLa-expressed NLpoly over the course of the evolution process with an overall  ̃105 improvement (from NLpolyWT:NLpepWT to NLpoly11S:NLpep80).
FIG. 102 shows the improvement in luminescence from HEK293 cell-expressed NLpoly over the course of the evolution process with an overall  ̃104 improvement (from NLpolyWT:NLpepWT to NLpoly11S:NLpep80).
FIG. 103 shows dissociation constants and demonstrates a  ̃104 fold improvement in binding affinity from NLpolyWT:NLpepWT to NLpoly11S:NLpep86.
FIG. 104 shows an image of a protein gel of total lysates and the soluble fraction of the same lysate for NLpoly variants from various steps of the evolution process.
FIG. 105 shows luminescence of various NLpolys in the absence of NLpep and in the presence of NLpep78 and NLpep79.
FIG. 106 shows luminescence of various NLpolys in the absence of NLpep and in the presence of NLpep78 and NLpep79.
FIG. 107 shows luminescence of various NLpolys in the absence of NLpep and in the presence of NLpep78 and NLpep79.
FIG. 108 shows a comparison of luminescence generated by cells expressing different combinations of FRB and FKBP fused to NLpolySP and NLpep80/87 after 15 min treatment with rapamycin or vehicle. Fold induction refers to signal generated in the presence of rapamycin compared to signal generated with vehicle.
FIG. 109 shows a comparison of luminescence generated by cells expressing different combinations of FRB and FKBP fused to NLpolySP and NLpep80/87 after 60 min treatment with rapamycin or vehicle.
FIG. 110 shows a comparison of luminescence generated by cells expressing different combinations of FRB and FKBP fused to NLpolySP and NLpep80/87 after 120 min treatment with rapamycin or vehicle.
FIG. 111 shows a comparison of luminescence generated by cells expressing different combinations of FRB and FKBP fused to NLpolySP and NLpep80/87 after 120 min treatment with rapamycin or vehicle. All 8 possible combinations of FRB and FKBP fused to NLpoly/NLpep were tested and less total DNA was used.
FIG. 112 shows a comparison of luminescence generated by FRB or FKBP fusions expressed in the absence of binding partner.
FIG. 113 shows a comparison of luminescence generated by cells transfected with varying amounts of FRB-NLpolySP and FKBP-NLpep80/87 DNA.
FIG. 114 shows a comparison of luminescence generated by cells transfected with varying amounts of FRB-NLpolySP or FKBP-NLpep80/87 DNA in the absence of binding partner.
FIG. 115 shows a comparison of luminescence generated by cells transfected with varying amounts of FRB-NLpolySP and FKBP-NLpep80/87 DNA. This example differs from FIG. 113 in that lower levels of DNA were used.
FIG. 116 shows a comparison of luminescence generated by cells transfected with varying amounts of FRB-NLpolySP or FKBP-NLpep80/87 DNA in the absence of binding partner. This differs from FIG. 114 in that lower levels of DNA were used.
FIG. 117 shows a comparison of luminescence generated by cells transfected with varying amounts of FRB-NLpolySP and FKBP-NLpep80 DNA after treatment with rapamycin for different lengths of time.
FIG. 118 shows a comparison of luminescence generated by cells transfected with varying amounts of FRB-NLpolySP and FKBP-NLpep87 DNA after treatment with rapamycin for different lengths of time.
FIG. 119 shows a comparison of luminescence generated by cells expressing different combinations of FRB-NLpoly5P with FKBP-NLpep80/87/95/96/97. Assay was performed in both a two-day and three-day format.
FIG. 120 shows a comparison of luminescence generated by cells expressing different combinations of FRB-NLpoly5A2 with FKBP-NLpep80/87/95/96/97. Assay was performed in both a two-day and three-day format.
FIG. 121 shows a comparison of luminescence generated by cells expressing different combinations of FRB-NLpoly5A2 or FRB-NLpoly11S with FKBP-NLpep101/104/105/106/107/108/109/110.
FIG. 122 shows a comparison of luminescence generated by cells transfected with different combinations of FRB-NLpoly5A2 or FRB-NLpoly11S with FKBP-NLpep87/96/98/99/100/101/102/103.
FIG. 123 shows a comparison of luminescence generated by cells transfected with different levels of FRB-NLpoly11S and FKBP-NLpep87/101/102/107 DNA.
FIG. 124 shows a comparison of luminescence generated by cells transfected with different levels of FRB-NLpoly5A2 and FKBP-NLpep87/101/102/107 DNA.
FIG. 125 shows a rapamycin dose response curve showing luminescence of cells expressing FRB-NLpoly5P and FKBP-NLpep80/87 DNA.
FIG. 126 shows a rapamycin dose response curve showing luminescence of cells expressing FRB-NLpoly5A2 or FRB-NLpoly11S and FKBP-NLpep87/101 DNA.
FIG. 127 shows a comparison of luminescence generated by cells expressing FRB-11S and FKBP-101 and treated with substrate PBI-4377 or furimazine.
FIG. 128 shows a rapamycin time course of cells expressing FRB-NLpoly11S/5A2 and FKBP-NLpep87/101 conducted in the presence or absence of rapamycin wherein the rapamycin was added manually.
FIG. 129 shows a rapamycin time course of cells expressing FRB-NLpoly11S/5A2 and FKBP-NLpep87/101 conducted in the presence or absence of rapamycin wherein the rapamycin was added via instrument injector.
FIG. 130 shows luminescence generated by FRB-NLpoly11S and FKBP-NLpep101 as measured on two different luminescence-reading instruments.
FIG. 131 provides images showing luminescence of cells expressing FRB-NLpoly11S and FKBP-NLpep101 at various times after treatment with rapamycin.
FIG. 132 provides a graph showing Image J quantitation of the signal generated by individual cells expressing FRB-NLpoly11S and FKBP-NLpep101 at various times after treatment with rapamycin.
FIG. 133 shows a comparison of luminescence in different cell lines expressing FRB-NLpoly 11S and FKBP-NLpep101.
FIG. 134 shows a comparison of luminescence generated by cells expressing FRB-NLpoly11S and FKBP-NLpep101 after treatment with the rapamycin competitive inhibitor FK506.
FIG. 135 shows (left side) luminescence generated by cells expressing FRB-NLpoly11S and FKBP-NLpep101 after treatment with the rapamycin competitive inhibitor FK506, and (right side) the percent of luminescence remaining after treatment with FK506.
FIG. 136 shows luminescence generated by cells transfected with different combinations of V2R-NLpoly5A2 or V2R-NLpoly11S with NLpep87/101-ARRB2 in the presence or absence of the V2R agonist AVP.
FIG. 137 shows an AVP treatment time course showing luminescence generated by cells transfected with V2R-NLpoly11S and NLpep87/101-ARRB2 after treatment with AVP wherein AVP was added manually.
FIG. 138 shows an AVP treatment time course showing luminescence generated by cells transfected with different combinations of V2R-NLpoly5A2 or V2R-NLpoly11S with NLpep87/101-ARRB2 after treatment with AVP wherein AVP was added via instrument injector.
FIG. 139 shows an AVP treatment time course at 37° C. showing luminescence generated by cells expressing different configurations of V2R and ARRB2 fused to NLpoly11S and NLpep101 after treatment with AVP.
FIG. 140 shows a comparison of luminescence in different cell lines expressing V2R-NLpep11S and NLpep101-ARRB2.
FIG. 141 shows 60× images showing luminescence of cells expressing V2R-NLpoly11S and NLpep101-ARRB2 at various times after treatment with AVP.
FIG. 142 shows 150× images showing luminescence of cells expressing V2R-NLpoly11S and NLpep101-ARRB2 at various times after treatment with AVP.
FIG. 143 shows a protein gel of total lysates and the soluble fraction of the same lysate for NLpoly variants.
FIG. 144 shows the dissociation constants for NLpoly 5P and combinations of mutations at positions 31, 46, 75, 76, and 93 in NLpoly 5P.
FIG. 145 shows a transferase example of post translational modification enzyme activity detection using an NLpep and aminopeptidase.
FIG. 146 shows a hydrolase example of post translational modification enzyme activity detection using an NLpep and methyl-specific antibody.
FIG. 147 contains wavelength scans for NLpoly WT complemented with either NLpepWT or NLpepWT conjugated to TMR.
FIG. 148 contains wavelength scans for NanoLuc fused to HaloTag (NL-HT) and NLpoly 5A2 complemented with NLPepWT with 4 additional amino acids (DEVD) and conjugated to Non-chloroTOM (NCT) (SEQ ID NO: 2579).
FIG. 149 shows a schematic a tertiary interaction wherein the energy transfer with an NLpoly and NLpep can also be used to measure three molecules interacting. In the schematic, a GPCR labeled with an NLpoly and a GPCR interacting protein labeled with an NLpep form a bioluminescent complex when they interact. This allows measurement of the binary interaction. If a small molecule GPCR ligand bearing an appropriate fluorescent moiety for energy transfer interacts with this system, energy transfer will occur. Therefore, the binary protein-protein interaction and the ternary drug-protein-protein interaction can be measured in the same experiment.
FIG. 150 shows a graph and table of binding affinities of NLpoly11S to synthetic NLPep78 and NLPep78 at the N- or C-terminus of a fusion partner (HaloTag).
FIG. 151 shows a graph and table of binding affinities of NLpoly11S to synthetic NLPep79 and NLPep79 at the N- or C-terminus of a fusion partner (HaloTag).
FIG. 152 shows a graph depicting normalized fluorescence intensity of NLpoly11S with NLPep86 or PBI-4877.
FIG. 153 shows a graph depicting normalized fluorescence intensity of NLpoly11S with NLPep86 or PBI-5434.
FIG. 154 shows a graph depicting normalized fluorescence intensity of NLpoly11S with NLPep86 or PBI-5436.
FIG. 155 shows a graph demonstrating furimazine binding affinity in affinity buffer of complexes between NLpoly11S and NLpep86, 78, 99, 101, 104, 128 and 114.
FIG. 156 shows a graph demonstrating furimazine binding affinity in NanoGlo assay buffer of complexes between NLpoly11S and NLpep86, 78, 99, 101, 104, 128 and 114.
FIG. 157 shows graphs depicting the change in affinity (NLpoly156/NLPep1 and NLpoly11S/NLPep1) with increasing concentrations of furimazine substrate.
FIG. 158 shows graphs depicting the change in affinity (NLpoly156/NLPep1 and NLpoly11S/NLPep1) with increasing concentrations of NLPep1.
FIG. 159 shows a graph depicting Vmax and Bmax NLPoly156, NLPoly11S, and NanoLuc® luciferase (Nluc) with NLPep1.
FIG. 160 shows a graph depicting RLU as a function of NLPep concentration for NLPoly11S and NLPep86, 78, 79, 99, 101, 104, 114, 128 and wt.
FIG. 161 shows a Western blot depicting expression level in HEK293T cells of NLPoly156 and NLPoly11S compared to full-length NanoLuc® luciferase.
FIG. 162 shows graphs depicting a comparison of the affinity of the β-lactamase SME and its inhibitor BLIPY50A as unfused proteins or when fused to NLPoly11S and NLPep114.
FIG. 163 shows a comparison of luminescence generated by cells expressing different combinations of FRB-NLpoly11S with FKBP-NLpep101/111-136
FIG. 164 shows a comparison of luminescence generated by cells expressing different combinations of FRB-NLpoly11S with FKBP-NLpep114 and 137-143.
FIG. 165 shows rapamycin dose response curves of cells expressing FRB-NLpoly11S and FKBP-NLpep78/79/99/101/104/114/128
FIG. 166 shows response of cells expressing FRB-NLpoly11S and FKBP-78/79/99/101/104/114/128 to the rapamycin competitive inhibitor FK506
FIG. 167 shows a comparison of luminescence generated by cells transfected with different ratios of FRB-NLpoly11S and FKBP-NLpep114.
FIG. 168 shows a comparison of luminescence generated by cells expressing NLpoly11S/NLpep114 fusions of FRB/FKBP in different orientations and with different linker lengths.
FIG. 169 shows graphs depicting rapamycin (A) dose-specific and (B) time-specific induction of FRB-NLpoly11S/FKBP-NLpep114 or split firefly complementation signals.
FIG. 170 shows graphs depicting FK506(A) dose-specific and (B) time-specific inhibition of FRB-NLpoly11S/FKBP-NLpep114 or split firefly complementation signals.
FIG. 171 shows Western blots depicting similar expression levels of FKBP-NLpep114 and FKBP-Fluc(394-544) at equal levels of transfected DNA.
FIG. 172 shows graphs depicting (A) dose-specific and (B) time-specific inhibition of NLpoly11S-BRD4 and Histone H3.3-NLpep114 interaction by IBET-151.
FIG. 173 shows a graph depicting dose dependent increases in RAS/CRAF, BRAF/BRAF and CRAF/BRAF dimerization in response to BRAF inhibitor GDC0879.
FIG. 174 shows a graph depicting RLU as a function of NLPep concentration for NLpoly11S and NLpep86 (SEQ ID NO: 390), wt (SEQ ID NO: 2578), and NLpep114 (SEQ ID NO: 2271).
FIG. 175 shows a schematic of an assay utilizing a high affinity peptide of a luminescent pair as an intracellular protein tag and the polypeptide of the luminescent pair as a detection reagent.
FIG. 176 shows a graph demonstrating the linear range of the affinity of NLpoly11S and MLpep86.
FIG. 177 shows images demonstrating the sensitivity of detecting proteins tagged with a high affinity NLPep using 11S. This figure also compares the detection using NLPep/NLPoly to the detection using fluorescently labeled HaloTag.
FIG. 178 shows a graph demonstrating the stability of NLpoly11S.
FIG. 179 shows a graph demonstrating the linear range of the affinity of NLpoly11S and NLpep78.
FIG. 180 shows a summary of NLpep sequences. High affinity (spontaneous) peptides are those peptides (NLpep) which bind to NLpoly11S with high affinity. Dark/Quencher peptides are those peptides (NLpep) which can reduce the levels of light being produced or detected from NLpoly11S.
FIG. 181 shows a schematic for the concept of structural complementation where the LSP and SSP (i.e., NLpoly and NLpep) are brought together to produce a bioluminescent signal (panels A, B). Upon disruption of a protein interaction (i.e. X and Y), LSP and SSP come apart resulting in a decrease in luminescence (Panel C).
FIGS. 182A-B show two options (A, B) for engineering structural complementation to be a loss of signal upon protein interaction between X and Y and a gain of signal upon disruption of the interaction between X and Y. Option A represents intermolecular structural complementation. Option B represents intramolecular structural complementation. FIG. 182B shows a list of genetic constructs that could be suitable for intramolecular structural complementation.
FIG. 183 shows (A) inhibition of NLpoly11S and NLpep114 binding by various dark peptides, and (B) dose-dependent inhibition by Lys-162 and Gln-162 peptides.
FIG. 184 A shows that inhibition by Q-162 and A-162 is dose-dependent. Panel B shows that Q-162 produces a signal on its own in a dose-dependent manner, while the dose-dependency of A-162 is subtle at best.
FIG. 185 shows graphs demonstrating dose-response of the dark peptides with CP Nluc.
FIG. 186 shows graphs depicting a time course of dark peptide with CP Nluc.
FIG. 187 shows the dark peptide dose-dependent inhibition of luminescence generated from FRB-NLpoly11S alone and also between FRB-NLpoly11S and FKBP-NLpep114 in the presence and absence of rapamycin.
FIG. 188 shows the dark peptide dose-dependent inhibition of luminescence generated from either FRB-NanoLuc (311) or NanoLuc-FRB (307) in the presence and absence of rapamycin (RLU).
FIG. 189 shows the dark peptide dose-dependent inhibition of luminescence generated from either FRB-NanoLuc (311) or NanoLuc-FRB (307) in the presence and absence of rapamycin (normalized to no dark peptide control; 100%).
FIG. 190 shows that the dark peptides, when fused to FKBP, can compete with both low (114) and high (80) affinity peptides (also FKBP fusions) and as a result reduce the total luminescence being produced and detected in live cells.
FIG. 191 shows the signal comparison between Fluc and NLpep86-based assays for intracellular levels of Fluc. The table depicts SEQ ID Nos: 172, 390, and 2581-2585.
FIG. 192 shows graphs demonstrating the utility of tandem linked NLpeps in complementing Npoly11S.
FIG. 193 shows a graph demonstrating that NLpoly and NLpep components do not interfere with intracellular degradation of reporter protein FlucP.
FIG. 194 shows a schematic demonstrating and extracellular protease activity assay.
FIG. 195 shows a schematic of an assay for measuring the activity of an enzyme using a ProNLpep.
FIG. 196 shows a schematic of an assay for screening antibodies, proteins, peptides or transporters that mediate cellular internalization.
FIG. 197 shows a schematic of a post-translational modification transferase assay.
FIG. 198 shows a schematic of a post-translational modification hydrolase assay.
FIG. 199 shows graphs correlating Tyrosine Kinase SRC activity with luminescence over background in a post-translational modification assay.
FIG. 200 shows a graph depicting spontaneous complementation of three different versions of NLpoly11S with twelve synthetic peptides.
FIG. 201 shows a schematic of a homogeneous immunoassay format utilizing fusions of NLpep and NLpoly with separate binding moieties A and B.
FIG. 202 shows graphs demonstrating: (A) reduction in background luminescence from NLpoly11S upon complex formation with GWALFKK (SEQ ID NO: 2351) and Dabcyl-GWALFKK (SEQ ID NO: 2351), and (B) NLpep86 forms a complex with NLpoly11S in the presence of GWALFKK (SEQ ID NO: 2351) and Dabcyl-GWALFKK (SEQ ID NO: 2351).
FIG. 203 shows graphs demonstrating: (A) VTGWALFEEIL (SEQ ID NO: 2372) (Trp 11mer) and VTGYALFEEIL (SEQ ID NO: 2355) (Tyr 11mer) induce luminescence over background (NLpoly11S alone; no peptide control), and that the N-terminal Dabcyl versions of each provide significant quenching of this signal, and (B) that NLpep86 forms a complex with NLpoly11S in the presence of Dabcyl versions of Trp 11mer and Tyr 11mer.



DEFINITIONS
As used herein, the term “substantially” means that the recited characteristic, parameter, and/or value need not be achieved exactly, but that deviations or variations, including for example, tolerances, measurement error, measurement accuracy limitations and other factors known to skill in the art, may occur in amounts that do not preclude the effect the characteristic was intended to provide. A characteristic or feature that is substantially absent (e.g., substantially non-luminescent) may be one that is within the noise, beneath background, below the detection capabilities of the assay being used, or a small fraction (e.g., <1%, <0.1%, <0.01%, <0.001%, <0.00001%, <0.000001%, <0.0000001%) of the significant characteristic (e.g., luminescent intensity of a bioluminescent protein or bioluminescent complex).
As used herein, the term “bioluminescence” refers to production and emission of light by a chemical reaction catalyzed by, or enabled by, an enzyme, protein, protein complex, or other biomolecule (e.g., bioluminescent complex). In typical embodiments, a substrate for a bioluminescent entity (e.g., bioluminescent protein or bioluminescent complex) is converted into an unstable form by the bioluminescent entity; the substrate subsequently emits light.
As used herein the term “complementary” refers to the characteristic of two or more structural elements (e.g., peptide, polypeptide, nucleic acid, small molecule, etc.) of being able to hybridize, dimerize, or otherwise form a complex with each other. For example, a “complementary peptide and polypeptide” are capable of coming together to form a complex. Complementary elements may require assistance to form a complex (e.g., from interaction elements), for example, to place the elements in the proper conformation for complementarity, to co-localize complementary elements, to lower interaction energy for complementary, etc.
As used herein, the term “complex” refers to an assemblage or aggregate of molecules (e.g., peptides, polypeptides, etc.) in direct and/or indirect contact with one another. In one aspect, “contact,” or more particularly, “direct contact” means two or more molecules are close enough so that attractive noncovalent interactions, such as Van der Waal forces, hydrogen bonding, ionic and hydrophobic interactions, and the like, dominate the interaction of the molecules. In such an aspect, a complex of molecules (e.g., a peptide and polypeptide) is formed under assay conditions such that the complex is thermodynamically favored (e.g., compared to a non-aggregated, or non-complexed, state of its component molecules). As used herein the term “complex,” unless described as otherwise, refers to the assemblage of two or more molecules (e.g., peptides, polypeptides or a combination thereof).
As used herein, the term “non-luminescent” refers to an entity (e.g., peptide, polypeptide, complex, protein, etc.) that exhibits the characteristic of not emitting a detectable amount of light in the visible spectrum (e.g., in the presence of a substrate). For example, an entity may be referred to as non-luminescent if it does not exhibit detectable luminescence in a given assay. As used herein, the term “non-luminescent” is synonymous with the term “substantially non-luminescent. For example, a non-luminescent polypeptide (NLpoly) is substantially non-luminescent, exhibiting, for example, a 10-fold or more (e.g., 100-fold, 200-fold, 500-fold, 1×103-fold, 1×104-fold, 1×105-fold, 1×106-fold, 1×107-fold, etc.) reduction in luminescence compared to a complex of the NLpoly with its non-luminescent complement peptide. In some embodiments, an entity is “non-luminescent” if any light emission is sufficiently minimal so as not to create interfering background for a particular assay.
As used herein, the terms “non-luminescent peptide” (e.g., NLpep) and “non-luminescent polypeptide” (e.g., NLpoly) refer to peptides and polypeptides that exhibit substantially no luminescence (e.g., in the presence of a substrate), or an amount that is beneath the noise, or a 10-fold or more (e.g., 100-fold, 200-fold, 500-fold, 1×103-fold, 1×104-fold, 1×105-fold, 1×106-fold, 1×107-fold, etc.) when compared to a significant signal (e.g., luminescent complex) under standard conditions (e.g., physiological conditions, assay conditions, etc.) and with typical instrumentation (e.g., luminometer, etc.). In some embodiments, such non-luminescent peptides and polypeptides assemble, according to the criteria described herein, to form a bioluminescent complex. As used herein, a “non-luminescent element” is a non-luminescent peptide or non-luminescent polypeptide. The term “bioluminescent complex” refers to the assembled complex of two or more non-luminescent peptides and/or non-luminescent polypeptides. The bioluminescent complex catalyzes or enables the conversion of a substrate for the bioluminescent complex into an unstable form; the substrate subsequently emits light. When uncomplexed, two non-luminescent elements that form a bioluminescent complex may be referred to as a “non-luminescent pair.” If a bioluminescent complex is formed by three or more non-luminescent peptides and/or non-luminescent polypeptides, the uncomplexed constituents of the bioluminescent complex may be referred to as a “non-luminescent group.”
As used herein, the term “interaction element” refers to a moiety that assists in bringing together a pair of non-luminescent elements or a non-luminescent group to form a bioluminescent complex. In a typical embodiment, a pair of interaction elements (a.k.a. “interaction pair”) is attached to a pair of non-luminescent elements (e.g., non-luminescent peptide/polypeptide pair), and the attractive interaction between the two interaction elements facilitates formation of the bioluminescent complex; although the present invention is not limited to such a mechanism, and an understanding of the mechanism is not required to practice the invention. Interaction elements may facilitate formation of the bioluminescent complex by any suitable mechanism (e.g., bringing non-luminescent pair/group into close proximity, placing a non-luminescent pair/group in proper conformation for stable interaction, reducing activation energy for complex formation, combinations thereof, etc.). An interaction element may be a protein, polypeptide, peptide, small molecule, cofactor, nucleic acid, lipid, carbohydrate, antibody, etc. An interaction pair may be made of two of the same interaction elements (i.e. homopair) or two different interaction elements (i.e. heteropair). In the case of a heteropair, the interaction elements may be the same type of moiety (e.g., polypeptides) or may be two different types of moieties (e.g., polypeptide and small molecule). In some embodiments, in which complex formation by the interaction pair is studied, an interaction pair may be referred to as a “target pair” or a “pair of interest,” and the individual interaction elements are referred to as “target elements” (e.g., “target peptide,” “target polypeptide,” etc.) or “elements of interest” (e.g., “peptide of interest,” “polypeptide or interest,” etc.).
As used herein, the term “preexisting protein” refers to an amino acid sequence that was in physical existence prior to a certain event or date. A “peptide that is not a fragment of a preexisting protein” is a short amino acid chain that is not a fragment or sub-sequence of a protein (e.g., synthetic or naturally-occurring) that was in physical existence prior to the design and/or synthesis of the peptide.
As used herein, the term “fragment” refers to a peptide or polypeptide that results from dissection or “fragmentation” of a larger whole entity (e.g., protein, polypeptide, enzyme, etc.), or a peptide or polypeptide prepared to have the same sequence as such. Therefore, a fragment is a subsequence of the whole entity (e.g., protein, polypeptide, enzyme, etc.) from which it is made and/or designed. A peptide or polypeptide that is not a subsequence of a preexisting whole protein is not a fragment (e.g., not a fragment of a preexisting protein). A peptide or polypeptide that is “not a fragment of a preexisting bioluminescent protein” is an amino acid chain that is not a subsequence of a protein (e.g., natural or synthetic) that: (1) was in physical existence prior to design and/or synthesis of the peptide or polypeptide, and (2) exhibits substantial bioluminescent activity.
As used herein, the term “subsequence” refers to peptide or polypeptide that has 100% sequence identify with another, larger peptide or polypeptide. The subsequence is a perfect sequence match for a portion of the larger amino acid chain.
As used herein, the term “sequence identity” refers to the degree two polymer sequences (e.g., peptide, polypeptide, nucleic acid, etc.) have the same sequential composition of monomer subunits. The term “sequence similarity” refers to the degree with which two polymer sequences (e.g., peptide, polypeptide, nucleic acid, etc.) have similar polymer sequences. For example, similar amino acids are those that share the same biophysical characteristics and can be grouped into the families, e.g., acidic (e.g., aspartate, glutamate), basic (e.g., lysine, arginine, histidine), non-polar (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan) and uncharged polar (e.g., glycine, asparagine, glutamine, cysteine, serine, threonine, tyrosine). The “percent sequence identity” (or “percent sequence similarity”) is calculated by: (1) comparing two optimally aligned sequences over a window of comparison (e.g., the length of the longer sequence, the length of the shorter sequence, a specified window), (2) determining the number of positions containing identical (or similar) monomers (e.g., same amino acids occurs in both sequences, similar amino acid occurs in both sequences) to yield the number of matched positions, (3) dividing the number of matched positions by the total number of positions in the comparison window (e.g., the length of the longer sequence, the length of the shorter sequence, a specified window), and (4) multiplying the result by 100 to yield the percent sequence identity or percent sequence similarity. For example, if peptides A and B are both 20 amino acids in length and have identical amino acids at all but 1 position, then peptide A and peptide B have 95% sequence identity. If the amino acids at the non-identical position shared the same biophysical characteristics (e.g., both were acidic), then peptide A and peptide B would have 100% sequence similarity. As another example, if peptide C is 20 amino acids in length and peptide D is 15 amino acids in length, and 14 out of 15 amino acids in peptide D are identical to those of a portion of peptide C, then peptides C and D have 70% sequence identity, but peptide D has 93.3% sequence identity to an optimal comparison window of peptide C. For the purpose of calculating “percent sequence identity” (or “percent sequence similarity”) herein, any gaps in aligned sequences are treated as mismatches at that position.
As used herein, the term “physiological conditions” encompasses any conditions compatible with living cells, e.g., predominantly aqueous conditions of a temperature, pH, salinity, chemical makeup, etc. that are compatible with living cells.
As used herein, the term “sample” is used in its broadest sense. In one sense, it is meant to include a specimen or culture obtained from any source, as well as biological and environmental samples. Biological samples may be obtained from animals (including humans) and encompass fluids, solids, tissues, and gases. Biological samples include blood products, such as plasma, serum and the like. Sample may also refer to cell lysates or purified forms of the peptides and/or polypeptides described herein. Cell lysates may include cells that have been lysed with a lysing agent or lysates such as rabbit reticulocyte or wheat germ lysates. Sample may also include cell-free expression systems. Environmental samples include environmental material such as surface matter, soil, water, crystals and industrial samples. Such examples are not however to be construed as limiting the sample types applicable to the present invention.
As used herein, unless otherwise specified, the terms “peptide” and “polypeptide” refer to polymer compounds of two or more amino acids joined through the main chain by peptide amide bonds (—C(O)NH—). The term “peptide” typically refers to short amino acid polymers (e.g., chains having fewer than 25 amino acids), whereas the term “polypeptide” typically refers to longer amino acid polymers (e.g., chains having more than 25 amino acids).
DETAILED DESCRIPTION
The study of protein interactions, particularly under physiological conditions and/or at physiologic expression levels, requires high sensitivity. In particular embodiments described herein, protein interactions with small molecules, nucleic acids, other proteins, etc. are detected based on the association of two non-luminescent elements that come together to from a bioluminescent complex capable of producing a detectable signal (e.g., luminescence). The formation of the bioluminescent complex is dependent upon the protein interaction that is being monitored.
Provided herein are compositions and methods for the assembly of a bioluminescent complex from two or more non-luminescent peptide and/or polypeptide units (e.g., non-luminescent pair). In some embodiments, the non-luminescent peptide and/or polypeptide units are not fragments of a preexisting protein (e.g., are not complementary subsequences of a known polypeptide sequence). In particular, bioluminescent activity is conferred upon a non-luminescent polypeptide via structural complementation with a non-luminescent peptide.
In some embodiments, provided herein are non-luminescent pairs for use in detecting and monitoring molecular interactions (e.g., protein-protein, protein-DNA, protein-RNA interactions, RNA-DNA, protein-small molecule, RNA-small-molecule, etc.). Also provided herein are complementary panels of interchangeable non-luminescent elements (e.g., peptides and polypeptides) that have variable affinities and luminescence upon formation of the various bioluminescent complexes (e.g., a high-affinity/high-luminescence pair, a moderate-affinity/high-luminescence pair, a low-affinity/moderate-luminescence pair, etc.). Utilizing different combinations of non-luminescent elements provides an adaptable system comprising various pairs ranging from lower to higher affinities, luminescence and other variable characteristics. This adaptability allows the detection/monitoring of molecular interactions to be fine-tuned to the specific molecule(s) of interest and expands the range of molecular interactions that can be monitored to include interactions with very high or low affinities. Further provided herein are methods by which non-luminescent pairs (or groups) and panels of non-luminescent pairs (or groups) are developed and tested.
In some embodiments, the interaction between the peptide/polypeptide members of the non-luminescent pair alone is insufficient to form the bioluminescent complex and produce the resulting bioluminescent signal. However, if an interaction element is attached to each peptide/polypeptide member of the non-luminescent pair, then the interactions of the interaction pair (e.g., to form an interaction complex) facilitate formation of the bioluminescent complex. In such embodiments, the bioluminescent signal from the bioluminescent complex (or the capacity to produce such a signal in the presence of substrate) serves as a reporter for the formation of the interaction complex. If an interaction complex is formed, then a bioluminescent complex is formed, and a bioluminescent signal is detected/measured/monitored (e.g., in the presence of substrate). If an interaction complex fails to form (e.g., due to unfavorable conditions, due to unstable interaction between the interaction elements, due to incompatible interaction elements), then a bioluminescent complex does not form, and a bioluminescent signal is not produced.
In certain embodiments, the interaction pair comprises two molecules of interest (e.g., proteins of interest). For example, assays can be performed to detect the interaction of two molecules of interest by tethering each one to a separate member of a non-luminescent pair. If the molecules of interest interact (e.g., transiently interact, stably interact, etc.), the non-luminescent pair is brought into close proximity in a suitable conformation and a bioluminescent complex is formed (and bioluminescent signal is produced/detected (in the presence of substrate)). In the absence of an interaction between the molecules of interest (e.g., no complex formation, not even transient interaction, etc.), the non-luminescent pair does not interact in a sufficient manner, and a bioluminescent signal is not produced or only weakly produced. Such embodiments can be used to study the effect of inhibitors on complex formation, the effect of mutations on complex formation, the effect of conditions (e.g., temperature, pH, etc.) on complex formation, the interaction of a small molecule (e.g., potential therapeutic) with a target molecule, etc.
Different non-luminescent pairs may require different strength, duration and/or stability of the interaction complex to result in bioluminescent complex formation. In some embodiments, a stable interaction complex is required to produce a detectable bioluminescent signal. In other embodiments, even a weak or transient interaction complex results in bioluminescent complex formation. In some embodiments, the strength or extent of an interaction complex is directly proportional to the strength of the resulting bioluminescent signal. Some non-luminescent pairs produce a detectable signal when combined with an interaction complex with a high millimolar dissociation constant (e.g., Kd>100 mM). Other non-luminescent pairs require an interaction pair with a low millimolar (e.g., Kd<100 mM), micromolar (e.g., Kd<1 mM), nanomolar (e.g., Kd<1 μM), or even picomolar (e.g., Kd<1 nM) dissociation constant in order to produce a bioluminescent complex with a detectable signal.
In some embodiments, one or more of the non-luminescent peptides/polypeptides are not fragments of a pre-existing protein. In some embodiments, one or more of the non-luminescent peptides/polypeptides are not fragments of a pre-existing bioluminescent protein. In some embodiments, neither/none of the non-luminescent peptides/polypeptides are fragments of a pre-existing protein. In some embodiments, neither/none of the non-luminescent peptides/polypeptides are fragments of a pre-existing bioluminescent protein. In some embodiments, neither the non-luminescent peptide nor non-luminescent polypeptide that assemble together to form a bioluminescent complex are fragments of a pre-existing protein. In some embodiments, a non-luminescent element for use in embodiments of the present invention is not a subsequence of a preexisting protein. In some embodiments, a non-luminescent pair for use in embodiments described herein does not comprise complementary subsequences of a preexisting protein.
In some embodiments, non-luminescent peptides/polypeptides are substantially non-luminescent in isolation. In certain embodiments, when placed in suitable conditions (e.g., physiological conditions), non-luminescent peptides/polypeptides interact to form a bioluminescent complex and produce a bioluminescent signal in the presence of substrate. In other embodiments, without the addition of one or more interaction elements (e.g., complementary interaction elements attached to the component non-luminescent peptide and non-luminescent polypeptide), non-luminescent peptides/polypeptides are unable to form a bioluminescent complex or only weakly form a complex. In such embodiments, non-luminescent peptides/polypeptides are substantially non-luminescent in each other's presence alone, but produce significant detectable luminescence when aggregated, associated, oriented, or otherwise brought together by interaction elements. In some embodiments, without the addition of one or more interaction elements (e.g., complementary interaction elements attached to the component peptide and polypeptide), peptides and/or polypeptides that assemble into the bioluminescent complex produce a low level of luminescence in each other's presence, but undergo a significant increase in detectable luminescence when aggregated, associated, oriented, or otherwise brought together by interaction elements.
In some embodiments, compositions and methods described herein comprise one or more interaction elements. In a typical embodiment, an interaction element is a moiety (e.g., peptide, polypeptide, protein, small molecule, nucleic acid, lipid, carbohydrate, etc.) that is attached to a peptide and/or polypeptide to assemble into the bioluminescent complex. The interaction element facilitates the formation of a bioluminescent complex by any suitable mechanism, including: interacting with one or both non-luminescent elements, inducing a conformational change in a non-luminescent element, interacting with another interaction element (e.g., an interaction element attached to the other non-luminescent element), bringing non-luminescent elements into close proximity, orienting non-luminescent elements for proper interaction, etc.
In some embodiments, one or more interaction elements are added to a solution containing the non-luminescent elements, but are not attached to the non-luminescent elements. In such embodiments, the interaction element(s) interact with the non-luminescent elements to induce formation of the bioluminescent complex or create conditions suitable for formation of the bioluminescent complex. In other embodiments, a single interaction element is attached to one member of a non-luminescent pair. In such embodiments, the lone interaction element interacts with one or both of the non-luminescent elements to create favorable interactions for formation of the bioluminescent complex. In typical embodiments of the present invention, one interaction element is attached to each member of a non-luminescent pair. Favorable interactions between the interaction elements facilitate interactions between the non-luminescent elements. The interaction pair may stably interact, transiently interact, form a complex, etc. The interaction of the interaction pair facilitates interaction of the non-luminescent elements (and formation of a bioluminescent complex) by any suitable mechanism, including, but not limited to: bringing the non-luminescent pair members into close proximity, properly orienting the non-luminescent pair members from interaction, reducing non-covalent forces acting against non-luminescent pair interaction, etc.
In some embodiments, an interaction pair comprises any two chemical moieties that facilitate interaction of an associated non-luminescent pair. An interaction pair may consist of, for example: two complementary nucleic acids, two polypeptides capable of dimerization (e.g., homodimer, heterodimer, etc.), a protein and ligand, protein and small molecule, an antibody and epitope, a reactive pair of small molecules, etc. Any suitable pair of interacting molecules may find use as an interaction pair.
In some embodiments, an interaction pair comprises two molecules of interest (e.g., proteins of interest) or target molecules. In some embodiments, compositions and methods herein provide useful assays (e.g., in vitro, in vivo, in situ, whole animal, etc.) for studying the interactions between a pair of target molecules.
In certain embodiments, a pair off interaction elements, each attached to one of the non-luminescent elements, interact with each other and thereby facilitate formation of the bioluminescent complex. In some embodiments, the presence of a ligand, substrate, co-factor or addition interaction element (e.g., not attached to non-luminescent element) is necessary to induce the interaction between the interaction elements and facilitate bioluminescent complex formation. In some embodiments, detecting a signal from the bioluminescent complex indicates the presence of the ligand, substrate, co-factor or addition interaction element or conditions that allow for interaction with the interaction elements.
In some embodiments, a pair off interaction elements, and a pair of non-luminescent elements are all present in a single amino acid chain (e.g., (interaction element 1)-NLpep-(interaction element 2)-NLpoly, NLpoly-(interaction element 1)-NLpep-(interaction element 2), NLpoly-(interaction element 1)-(interaction element 2)-NLpep, etc.). In some embodiments in which a pair off interaction elements, and a pair of non-luminescent elements are all present in a single amino acid chain, a ligand, substrate, co-factor or addition interaction element is required for the interaction pair to form an interaction complex and facilitate formation of the bioluminescent complex.
In certain embodiments, an interaction element and a non-luminescent element are attached, fused, linked, connected, etc. In typical embodiments, a first non-luminescent element and a first interaction element are attached to each other, and a second non-luminescent element and a second interaction element are attached to each other. Attachment of signal and interaction elements may be achieved by any suitable mechanism, chemistry, linker, etc. The interaction and non-luminescent elements are typically attached through covalent connection, but non-covalent linking of the two elements is also provided. In some embodiments, the signal and interaction elements are directly connected and, in other embodiments, they are connected by a linker.
In some embodiments, in which the interaction element is a peptide or polypeptide, the signal and interaction elements are contained within a single amino acid chain. In some embodiments, a single amino acid chain comprises, consists of, or consists essentially of a non-luminescent element and interaction element. In some embodiments, a single amino acid chain comprises, consists of, or consists essentially of a non-luminescent element, an interaction element, and optionally one or more an N-terminal sequence, a C-terminal sequence, regulatory elements (e.g., promoter, translational start site, etc.), and a linker sequence. In some embodiments, the signal and interaction elements are contained within a fusion polypeptide. The signal and interaction elements (and any other amino acid segments to be included in the fusion) may be expressed separately; however, in other embodiments, a fusion protein is expressed that comprises or consist of both the interaction and signal sequences.
In some embodiments, a first fusion protein comprising a first non-luminescent element and first interaction element as well as a second fusion protein comprising a second non-luminescent element and second interaction element are expressed within the same cells. In such embodiments, the first and second fusion proteins are purified and/or isolated from the cells, or the interaction of the fusion proteins is assayed within the cells. In other embodiments, first and second fusion proteins are expressed in separate cells and combined (e.g., following purification and/or isolation, or following fusion of the cells or portions of the cells, or by transfer of a fusion protein from one cell to another, or by secretion of one or more fusion proteins into the extracellular medium) for signal detection. In some embodiments, one or more fusion proteins are expressed in cell lysate (e.g., rabbit reticulocyte lysate) or in a cell-free system. In some embodiments, one or more fusion proteins are expressed from the genome of a virus or other cellular pathogen.
In certain embodiments, nucleic acids, DNA, RNA, vectors, etc. are provided that encode peptide, polypeptides, fusion polypeptide, fusion proteins, etc. of the present invention. Such nucleic acids and vectors may be used for expression, transformation, transfection, injection, etc.
In some embodiments, a non-luminescent element and interaction element are connected by a linker. In some embodiments, a linker connects the signal and interaction elements while providing a desired amount of space/distance between the elements. In some embodiments, a linker allows both the signal and interaction elements to form their respective pairs (e.g., non-luminescent pair and interaction pair) simultaneously. In some embodiments, a linker assists the interaction element in facilitating the formation of a non-luminescent pair interaction. In some embodiments, when an interaction pair is formed, the linkers that connect each non-luminescent element to their respective interaction elements position the non-luminescent elements at the proper distance and conformation to form a bioluminescent complex. In some embodiments, an interaction element and non-luminescent element are held in close proximity (e.g., <4 monomer units) by a linker. In some embodiments, a linker provides a desired amount of distance (e.g., 1, 2, 3, 4, 5, 6 . . . 10 . . . 20, or more monomer units) between signal and interaction elements (e.g., to prevent undesirable interactions between signal and interaction elements, for steric considerations, to allow proper orientation of non-luminescent element upon formation of interaction complex, to allow propagation of a complex-formation from interaction complex to non-luminescent elements, etc.). In certain embodiments, a linker provides appropriate attachment chemistry between the signal and interaction elements. A linker may also improve the synthetic process of making the signal and interaction element (e.g., allowing them to be synthesized as a single unit, allowing post synthesis connection of the two elements, etc.).
In some embodiments, a linker is any suitable chemical moiety capable of linking, connecting, or tethering a non-luminescent element to an interaction element. In some embodiments, a linker is a polymer of one or more repeating or non-repeating monomer units (e.g., nucleic acid, amino acid, carbon-containing polymer, carbon chain, etc.). When a non-luminescent element and interaction element are part of a fusion protein, a linker (when present) is typically an amino acid chain. When a non-luminescent element and interaction element are tethered together after the expression of the individual elements, a linker may comprise any chemical moiety with functional (or reactive) groups at either end that are reactive with functional groups on the signal and interaction elements, respectively. Any suitable moiety capable of tethering the signal and interaction elements may find use as a linker.
A wide variety of linkers may be used. In some embodiments, the linker is a single covalent bond. In some embodiments, the linker comprises a linear or branched, cyclic or heterocyclic, saturated or unsaturated, structure having 1-20 nonhydrogen atoms (e.g., C, N, P, O and S) and is composed of any combination of alkyl, ether, thioether, imine, carboxylic, amine, ester, carboxamide, sulfonamide, hydrazide bonds and aromatic or heteroaromatic bonds. In some embodiments, linkers are longer than 20 nonhydrogen atoms (e.g. 21 non-hydrogen atoms, 25 non-hydrogen atoms, 30 non-hydrogen atoms, 40 non-hydrogen atoms, 50 non-hydrogen atoms, 100 non-hydrogen atoms, etc.) In some embodiments, the linker comprises 1-50 non-hydrogen atoms (in addition to hydrogen atoms) selected from the group of C, N, P, O and S (e.g. 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50 non-hydrogen atoms).
The present invention is not limited by the types of linkers available. The signal and interaction elements are linked, either directly (e.g. linker consists of a single covalent bond) or linked via a suitable linker. The present invention is not limited to any particular linker group. A variety of linker groups are contemplated, and suitable linkers could comprise, but are not limited to, alkyl groups, methylene carbon chains, ether, polyether, alkyl amide linker, a peptide linker, a modified peptide linker, a Poly(ethylene glycol) (PEG) linker, a streptavidin-biotin or avidin-biotin linker, polyaminoacids (e.g. polylysine), functionalised PEG, polysaccharides, glycosaminoglycans, dendritic polymers (WO93/06868 and by Tomalia et al. in Angew. Chem. Int. Ed. Engl. 29:138-175 (1990), herein incorporated by reference in their entireties), PEG-chelant polymers (W94/08629, WO94/09056 and WO96/26754, herein incorporated by reference in their entireties), oligonucleotide linker, phospholipid derivatives, alkenyl chains, alkynyl chains, disulfide, or a combination thereof. In some embodiments, the linker is cleavable (e.g., enzymatically (e.g., TEV protease site), chemically, photoinduced, etc.
In some embodiments, substantially non-luminescent peptides and polypeptides are provided with less than 100% sequence identity and/or similarity to any portion of an existing luciferase (e.g., a firefly luciferase, a Renilla luciferase, an Oplophorus luciferase, enhanced Oplophorus luciferases as described in U.S. Pat. App. 2010/0281552 and U.S. Pat. App. 2012/0174242, herein incorporated by reference in their entireties). Certain embodiments of the present invention involve the formation of bioluminescent complexes of non-luminescent peptides and polypeptides with less than 100% sequence identity with all or a portion (e.g., 8 or more amino acids, less than about 25 amino acids for peptides) of SEQ ID NO: 2157 (e.g., complete NANOLUC sequence). Certain embodiments of the present invention involve the formation of bioluminescent complexes of non-luminescent peptides and polypeptides with less than 100%, but more than 40% (e.g., >40%, >45%, >50%, >55%, >60%, >65%, >70%, >75%, >80%, >85%, >90%, >95%, >98%, >99%) sequence identity with all or a portion (e.g., 8 or more amino acids, less than about 25 amino acids for peptides) of SEQ ID NO: 2157 (e.g., complete NANOLUC sequence). In some embodiments, non-luminescent peptides and polypeptides are provided with less than 100% sequence similarity with a portion (e.g., 8 or more amino acids, less than about 25 amino acids for peptides) of SEQ ID NO: 2157 (e.g., peptides and polypeptides that interact to form bioluminescent complexes). In some embodiments, non-luminescent peptides and polypeptides are provided with less than 100%, but more than 40% (e.g., >40%, >45%, >50%, >55%, >60%, >65%, >70%, >75%, >80%, >85%, >90%, >95%, >98%, >99%) sequence similarity with a portion (e.g., 8 or more amino acids, less than about 25 amino acids for peptides) of SEQ ID NO: 2157 (e.g., peptides and polypeptides that interact to form bioluminescent complexes). Non-luminescent peptides are provided that have less than 100% sequence identity and/or similarity with about a 25 amino acid or less portion of SEQ ID NO: 2157, wherein such peptides form a bioluminescent complex when combined under appropriate conditions (e.g., stabilized by an interaction pair) with a polypeptide having less than 100%, but more than 40% (e.g., >40%, >45%, >50%, >55%, >60%, >65%, >70%, >75%, >80%, >85%, >90%, >95%, >98%, >99%) sequence identity and/or similarity with another portion SEQ ID NO: 2157. Non-luminescent peptides are provided that have less than 100% sequence identity and/or similarity with about a 25 amino acid or less portion of SEQ ID NO: 2157, wherein such peptides form a bioluminescent complex when combined under appropriate conditions (e.g., stabilized by an interaction pair) with a polypeptide having less than 100%, but more than 40% (e.g., >40%, >45%, >50%, >55%, >60%, >65%, >70%, >75%, >80%, >85%, >90%, >95%, >98%, >99%) sequence identity and/or similarity with another portion SEQ ID NO: 2157. Non-luminescent peptides are provided that have less than 100%, but more than 40% (e.g., >40%, >45%, >50%, >55%, >60%, >65%, >70%, >75%, >80%, >85%, >90%, >95%, >98%, >99%) sequence identity and/or similarity with about a 25 amino acid or less portion of SEQ ID NO: 2157, wherein such peptides form a bioluminescent complex when combined under appropriate conditions (e.g., stabilized by an interaction pair) with a polypeptide having less than 100%, but more than 40% (e.g., >40%, >45%, >50%, >55%, >60%, >65%, >70%, >75%, >80%, >85%, >90%, >95%, >98%, >99%) sequence identity and/or similarity with another portion SEQ ID NO: 2157. Similarly, non-luminescent polypeptides are provided that have less than 100%, but more than 40% (e.g., >40%, >45%, >50%, >55%, >60%, >65%, >70%, >75%, >80%, >85%, >90%, >95%, >98%, >99%) sequence identity or similarity with a portion of SEQ ID NO: 2157, wherein such polypeptides form a bioluminescent complex when combined under appropriate conditions (e.g., stabilized by an interaction pair) with a peptide having less than 100%, but optionally more than 40% (e.g., >40%, >45%, >50%, >55%, >60%, >65%, >70%, >75%, >80%, >85%, >90%, >95%, >98%, >99%) sequence identity and/or similarity with another portion SEQ ID NO: 2157. In some embodiments, non-luminescent peptides with less than 100 sequence identity or similarity with SEQ ID NO: 2 are provided. In some embodiments, non-luminescent peptides with less than 100%, but more than 40% (e.g., >40%, >45%, >50%, >55%, >60%, >65%, >70%, >75%, >80%, >85%, >90%, >95%, >98%, >99%) sequence identity or similarity with SEQ ID NO: 2 are provided. In some embodiments, non-luminescent polypeptides with less than 100 sequence identity or similarity with SEQ ID NO: 440 are provided.
In some embodiments, non-luminescent polypeptides with less than 100%, but more than 40% (e.g., >40%, >45%, >50%, >55%, >60%, >65%, >70%, >75%, >80%, >85%, >90%, >95%, >98%, >99%) sequence identity or similarity with SEQ ID NO: 440 are provided.
In some embodiments, non-luminescent peptides that find use in embodiments of the present invention include peptides with one or more amino acid substitutions, deletions, or additions from GVTGWRLCKRISA (SEQ ID NO: 236). In some embodiments, the present invention provides peptides comprising one or more of amino acid sequences of Table 1, and/or nucleic acids comprising the nucleic acid sequences of Table 1 (which code for the peptide sequences of Table 1).







TABLE 1







Peptide sequences










SEQ





ID





NO.
PEPTIDE NO.
POLYMER
SEQUENCE













3
NLpep2 (w/ Met)
N.A.
ATGGACGTGACCGGCTGGCGGCTGTGCGAACGCATTCT





GGCG


 


4
NLpep2 (w/ Met)
A.A.
MDVTGWRLCERILA


 


5
NLpep3 (w/ Met)
N.A.
ATGGGAGTGACCGCCTGGCGGCTGTGCGAACGCATTCT





GGCG


 


6
NLpep3 (w/ Met)
A.A.
MGVTAWRLCERILA


 


7
NLpep4 (w/ Met)
N.A.
ATGGGAGTGACCGGCTGGCGGCTGTGCAAGCGCATTC





TGGCG


 


8
NLpep4 (w/ Met)
A.A.
MGVTGWRLCKRILA


 


9
NLpep5 (w/ Met)
N.A.
ATGGGAGTGACCGGCTGGCGGCTGTGCGAACGCATTA





GCGCG


 


10
NLpep5 (w/ Met)
A.A.
MGVTGWRLCERISA


 


11
NLpep6 (w/ Met)
N.A.
ATGGACGTGACCGGCTGGCGGCTGTGCAAGCGCATTA





GCGCG


 


12
NLpep6 (w/ Met)
A.A.
MDVTGWRLCKRISA


 


13
NLpep7 (w/ Met)
N.A.
ATGGACGTGACCGGCTGGCGGCTGTGCAAGCGCATTCT





GGCG


 


14
NLpep7 (w/ Met)
A.A.
MDVTGWRLCKRILA


 


15
NLpep8 (w/ Met)
N.A.
ATGGACGTGACCGGCTGGCGGCTGTGCGAACGCATTA





GCGCG


 


16
NLpep8 (w/ Met)
A.A.
MDVTGWRLCERISA


 


17
NLpep9 (w/ Met)
N.A.
ATGGGAGTGACCGGCTGGCGGCTGTGCAAGCGCATTA





GCGCG


 


18
NLpep9 (w/ Met)
A.A.
MGVTGWRLCKRISA


 


19
NLpep10 (w/ Met)
N.A.
ATGGGAGTGACCGGCTGGCGGCTGAACGAACGCATTC





TGGCG


 


20
NLpep10 (w/ Met)
A.A.
MGVTGWRLNERILA


 


21
NLpep11 (w/ Met)
N.A.
ATGGGAGTGACCGGCTGGCGGCTGCAGGAACGCATTC





TGGCG


 


22
NLpep11 (w/ Met)
A.A.
MGVTGWRLQERILA


 


23
NLpep12 (w/ Met)
N.A.
ATGGGAGTGACCGGCTGGCGGCTGAAGAAGCGCCGGA





GCCGG


 


24
NLpep12 (w/ Met)
A.A.
MGVTGWRLKKRRSR


 


25
NLpep13 (w/ Met)
N.A.
ATGAACGTGACCGGCTGGCGGCTGTGCAAGCGCATTA





GCGCG


 


26
NLpep13 (w/ Met)
A.A.
MNVTGWRLCKRISA


 


27
NLpep14 (w/ Met)
N.A.
ATGAGCGTGACCGGCTGGCGGCTGTGCAAGCGCATTA





GCGCG


 


28
NLpep14 (w/ Met)
A.A.
MSVTGWRLCKRISA


 


29
NLpep15 (w/ Met)
N.A.
ATGGAGGTGACCGGCTGGCGGCTGTGCAAGCGCATTA





GCGCG


 


30
NLpep15 (w/ Met)
A.A.
MEVTGWRLCKRISA


 


31
NLpep16 (w/ Met)
N.A.
ATGGGCGTGACCGGCTGGCGGCTGTGCAAGCGCATTA





GCGCG


 


32
NLpep16 (w/ Met)
A.A.
MHVTGWRLCKRISA


 


33
NLpep17 (w/ Met)
N.A.
ATGGGACACACCGGCTGGCGGCTGTGCAAGCGCATTA





GCGCG


 


34
NLpep17 (w/ Met)
A.A.
MGITGWRLCKRISA


 


35
NLpep18 (w/ Met)
N.A.
ATGGGAGCCACCGGCTGGCGGCTGTGCAAGCGCATTA





GCGCG


 


36
NLpep18 (w/ Met)
A.A.
MGATGWRLCKRISA


 


37
NLpep19 (w/ Met)
N.A.
ATGGGAAAGACCGGCTGGCGGCTGTGCAAGCGCATTA





GCGCG


 


38
NLpep19 (w/ Met)
A.A.
MGKTGWRLCKRISA


 


39
NLpep20 (w/ Met)
N.A.
ATGGGACAGACCGGCTGGCGGCTGTGCAAGCGCATTA





GCGCG


 


40
NLpep20 (w/ Met)
A.A.
MGQTGWRLCKRISA


 


41
NLpep21 (w/ Met)
N.A.
ATGGGAAGCACCGGCTGGCGGCTGTGCAAGCGCATTA





GCGCG


 


42
NLpep21 (w/ Met)
A.A.
MGSTGWRLCKRISA


 


43
NLpep22 (w/ Met)
N.A.
ATGGGAGTGGTGGGCTGGCGGCTGTGCAAGCGCATTA





GCGCG


 


44
NLpep22 (w/ Met)
A.A.
MGVVGWRLCKRISA


 


45
NLpep23 (w/ Met)
N.A.
ATGGGAGTGAAGGGCTGGCGGCTGTGCAAGCGCATTA





GCGCG


 


46
NLpep23 (w/ Met)
A.A.
MGVKGWRLCKRISA


 


47
NLpep24 (w/ Met)
N.A.
ATGGGAGTGCAGGGCTGGCGGCTGTGCAAGCGCATTA





GCGCG


 


48
NLpep24 (w/ Met)
A.A.
MGVQGWRLCKRISA


 


49
NLpep25 (w/ Met)
N.A.
ATGGGAGTGACCGGCACCCGGCTGTGCAAGCGCATTA





GCGCG


 


50
NLpep25 (w/ Met)
A.A.
MGVTGTRLCKRISA


 


51
NLpep26 (w/ Met)
N.A.
ATGGGAGTGACCGGCAAGCGGCTGTGCAAGCGCATTA





GCGCG


 


52
NLpep26 (w/ Met)
A.A.
MGVTGKRLCKRISA


 


53
NLpep27 (w/ Met)
N.A.
ATGGGAGTGACCGGCGTGCGGCTGTGCAAGCGCATTA





GCGCG


 


54
NLpep27 (w/ Met)
A.A.
MGVTGVRLCKRISA


 


55
NLpep28 (w/ Met)
N.A.
ATGGGAGTGACCGGCTGGCGGCACTGCAAGCGCATTA





GCGCG


 


56
NLpep28 (w/ Met)
A.A.
MGVTGWRICKRISA


 


57
NLpep29 (w/ Met)
N.A.
ATGGGAGTGACCGGCTGGCGGGTGTGCAAGCGCATTA





GCGCG


 


58
NLpep29 (w/ Met)
A.A.
MGVTGWRVCKRISA


 


59
NLpep30 (w/ Met)
N.A.
ATGGGAGTGACCGGCTGGCGGACCTGCAAGCGCATTA





GCGCG


 


60
NLpep30 (w/ Met)
A.A.
MGVTGWRTCKRISA


 


61
NLpep31 (w/ Met)
N.A.
ATGGGAGTGACCGGCTGGCGGTACTGCAAGCGCATTA





GCGCG


 


62
NLpep31 (w/ Met)
A.A.
MGVTGWRYCKRISA


 


63
NLpep32 (w/ Met)
N.A.
ATGGGAGTGACCGGCTGGCGGAAGTGCAAGCGCATTA





GCGCG


 


64
NLpep32 (w/ Met)
A.A.
MGVTGWRKCKRISA


 


65
NLpep33 (w/ Met)
N.A.
ATGGGAGTGACCGGCTGGCGGCTGAACAAGCGCATTA





GCGCG


 


66
NLpep33 (w/ Met)
A.A.
MGVTGWRLNKRISA


 


67
NLpep34 (w/ Met)
N.A.
ATGGGAGTGACCGGCTGGCGGCTGACCAAGCGCATTA





GCGCG


 


68
NLpep34 (w/ Met)
A.A.
MGVTGWRLTKRISA


 


69
NLpep35 (w/ Met)
N.A.
ATGGGAGTGACCGGCTGGCGGCTGTGCAAGAAGATTA





GCGCG


 


70
NLpep35 (w/ Met)
A.A.
MGVTGWRLCKKISA


 


71
NLpep36 (w/ Met)
N.A.
ATGGGAGTGACCGGCTGGCGGCTGTGCAAGAACATTA





GCGCG


 


72
NLpep36 (w/ Met)
A.A.
MGVTGWRLCKNISA


 


73
NLpep37 (w/ Met)
N.A.
ATGGGAGTGACCGGCTGGCGGCTGTGCAAGCGCGTGA





GCGCG


 


74
NLpep37 (w/ Met)
A.A.
MGVTGWRLCKRVSA


 


75
NLpep38 (w/ Met)
N.A.
ATGGGAGTGACCGGCTGGCGGCTGTGCAAGCGCCAGA





GCGCG


 


76
NLpep38 (w/ Met)
A.A.
MGVTGWRLCKRQSA


 


77
NLpep39 (w/ Met)
N.A.
ATGGGAGTGACCGGCTGGCGGCTGTGCAAGCGCGAGA





GCGCG


 


78
NLpep39 (w/ Met)
A.A.
MGVTGWRLCKRESA


 


79
NLpep40 (w/ Met)
N.A.
ATGGGAGTGACCGGCTGGCGGCTGTGCAAGCGCCGGA





GCGCG


 


80
NLpep40 (w/ Met)
A.A.
MGVTGWRLCKRRSA


 


81
NLpep41 (w/ Met)
N.A.
ATGGGAGTGACCGGCTGGCGGCTGTGCAAGCGCTTCA





GCGCG


 


82
NLpep41 (w/ Met)
A.A.
MGVTGWRLCKRFSA


 


83
NLpep42 (w/ Met)
N.A.
ATGGGAGTGACCGGCTGGCGGCTGTGCAAGCGCATTA





GCAAC


 


84
NLpep42 (w/ Met)
A.A.
MGVTGWRLCKRISN


 


85
NLpep43 (w/ Met)
N.A.
ATGGGAGTGACCGGCTGGCGGCTGTGCAAGCGCATTA





GCACC


 


86
NLpep43 (w/ Met)
A.A.
MGVTGWRLCKRIST


 


87
NLpep44 (w/ Met)
N.A.
ATGGGAGTGACCGGCTGGCGGCTGTGCAAGCGCATTA





GCCGG


 


88
NLpep44 (w/ Met)
A.A.
MGVTGWRLCKRISR


 


89
NLpep45 (w/ Met)
N.A.
ATGGGAGTGACCGGCTGGCGGCTGTGCAAGCGCATTA





GCCTG


 


90
NLpep45 (w/ Met)
A.A.
MGVTGWRLCKRISL


 


91
NLpep46 (w/ Met)
N.A.
ATGGGAGTGACCGGCTGGCGGCTGTGCAAGCGCATTA





GCGAG


 


92
NLpep46 (w/ Met)
A.A.
MGVTGWRLCKRISE


 


93
NLpep47 (w/ Met)
N.A.
ATGGGAGTGACCGGCTTCCGGCTGTGCAAGCGCATTA





GCGCG


 


94
NLpep47 (w/ Met)
A.A.
MGVTGFRLCKRISA


 


95
NLpep48 (w/ Met)
N.A.
ATGGGAGTGACCGGCTACCGGCTGTGCAAGCGCATTA





GCGCG


 


96
NLpep48 (w/ Met)
A.A.
MGVTGYRLCKRISA


 


97
NLpep49 (w/ Met)
N.A.
ATGGGAGTGACCGGCGAGCGGCTGTGCAAGCGCATTA





GCGCG


 


98
NLpep49 (w/ Met)
A.A.
MGVTGERLCKRISA


 


99
NLpep50 (w/ Met)
N.A.
ATGCAGGTGACCGGCTGGCGGCTGTGCAAGCGCATTA





GCGCG


 


100
NLpep50 (w/ Met)
A.A.
MQVTGWRLCKRISA


 


101
NLpep51 (w/ Met)
N.A.
ATGACCGTGACCGGCTGGCGGCTGTGCAAGCGCATTA





GCGCG


 


102
NLpep51 (w/ Met)
A.A.
MTVTGWRLCKRISA


 


103
NLpep52 (w/ Met)
N.A.
ATGGGAGTGGAGGGCTGGCGGCTGTGCAAGCGCATTA





GCGCG


 


104
NLpep52 (w/ Met)
A.A.
MGVEGWRLCKRISA


 


105
NLpep53 (w/ Met)
N.A.
ATGGGAGTGACCGGCTGGCGGCTGTTCAAGCGCATTA





GCGCG


 


106
NLpep53 (w/ Met)
A.A.
MGVTGWRLFKRISA


 


107
NLpep54 (w/ Met)
N.A.
ATGGGAGTGACCGGCTGGCGGCTGTACAAGCGCATTA





GCGCG


 


108
NLpep54 (w/ Met)
A.A.
MGVTGWRLYKRISA


 


109
NLpep55 (w/ Met)
N.A.
ATGGGAGTGACCGGCTGGCGGCTGAGCAAGCGCATTA





GCGCG


 


110
NLpep55 (w/ Met)
A.A.
MGVTGWRLSKRISA


 


111
NLpep56 (w/ Met)
N.A.
ATGGGAGTGACCGGCTGGCGGCTGGGCAAGCGCATTA





GCGCG


 


112
NLpep56 (w/ Met)
A.A.
MGVTGWRLHKRISA


 


113
NLpep57 (w/ Met)
N.A.
ATGGGAGTGACCGGCTGGCGGCTGATGAAGCGCATTA





GCGCG


 


114
NLpep57 (w/ Met)
A.A.
MGVTGWRLMKRISA


 


115
NLpep58 (w/ Met)
N.A.
ATGGGAGTGACCGGCTGGCGGCTGGCCAAGCGCATTA





GCGCG


 


116
NLpep58 (w/ Met)
A.A.
MGVTGWRLAKRISA


 


117
NLpep59 (w/ Met)
N.A.
ATGGGAGTGACCGGCTGGCGGCTGCAGAAGCGCATTA





GCGCG


 


118
NLpep59 (w/ Met)
A.A.
MGVTGWRLQKRISA


 


119
NLpep60 (w/ Met)
N.A.
ATGGGAGTGACCGGCTGGCGGCTGCTGAAGCGCATTA





GCGCG


 


120
NLpep60 (w/ Met)
A.A.
MGVTGWRLLKRISA


 


121
NLpep61 (w/ Met)
N.A.
ATGGGAGTGACCGGCTGGCGGCTGAAGAAGCGCATTA





GCGCG


 


122
NLpep61 (w/ Met)
A.A.
MGVTGWRLKKRISA


 


123
NLpep62 (w/ Met)
N.A.
ATGAACCACACCGGCTGGCGGCTGAACAAGAAGGTGA





GCAAC


 


124
NLpep62 (w/ Met)
A.A.
MNITGWRLNKKVSN


 


125
NLpep63 (w/ Met)
N.A.
ATGAACCACACCGGCTACCGGCTGAACAAGAAGGTGA





GCAAC


 


126
NLpep63 (w/ Met)
A.A.
MNITGYRLNKKVSN


 


127
NLpep64 (w/ Met)
N.A.
ATGTGCGTGACCGGCTGGCGGCTGTTCAAGCGCATTAG





CGCG


 


128
NLpep64 (w/ Met)
A.A.
MCVTGWRLFKRISA


 


129
NLpep65 (w/ Met)
N.A.
ATGCCCGTGACCGGCTGGCGGCTGTTCAAGCGCATTAG





CGCG


 


130
NLpep65 (w/ Met)
A.A.
MPVTGWRLFKRISA


 


131
NLpep66 (w/ Met)
N.A.
ATGAACCACACCGGCTACCGGCTGTTCAAGAAGGTGA





GCAAC


 


132
NLpep66 (w/ Met)
A.A.
MNITGYRLFKKVSN


 


133
NLpep67 (w/ Met)
N.A.
ATGAACGTGACCGGCTACCGGCTGTTCAAGAAGGTGA





GCAAC


 


134
NLpep67 (w/ Met)
A.A.
MNVTGYRLFKKVSN


 


135
NLpep68 (w/ Met)
N.A.
ATGAACGTGACCGGCTGGCGGCTGTTCAAGAAGGTGA





GCAAC


 


136
NLpep68 (w/ Met)
A.A.
MNVTGWRLFKKVSN


 


137
NLpep69 (w/ Met)
N.A.
ATGAACGTGACCGGCTGGCGGCTGTTCAAGAAGATTA





GCAAC


 


138
NLpep69 (w/ Met)
A.A.
MNVTGWRLFKKISN


 


139
NLpep70 (w/ Met)
N.A.
ATGAACGTGACCGGCTGGCGGCTGTTCAAGCGCATTA





GCAAC


 


140
NLpep70 (w/ Met)
A.A.
MNVTGWRLFKRILN


 


141
NLpep71 (w/ Met)
N.A.
ATGGGAGTGACCGGCTGGCGGCTGTTCAAGCGCATTA





GCAAC


 


142
NLpep71 (w/ Met)
A.A.
MGVTGWRLFKRISN


 


143
NLpep72 (w/ Met)
N.A.
ATGAACGTGACCGGCTGGCGGCTGTTCGAACGCATTA





GCAAC


 


144
NLpep72 (w/ Met)
A.A.
MNVTGWRLFERISN


 


145
NLpep73 (w/ Met)
N.A.
ATGAACGTGACCGGCTGGCGGCTGTTCAAGCGCATTCT





GAAC


 


146
NLpep73 (w/ Met)
A.A.
MNVTGWRLFKRILN


 


147
NLpep74 (w/ Met)
N.A.
ATGAACGTGACCGGCTGGCGGCTGTTCAAGCGCATTA





GCGCG


 


148
NLpep74 (w/ Met)
A.A.
MNVTGWRLFKRISA


 


149
NLpep75 (w/ Met)
N.A.
ATGAACGTGACCGGCTGGCGGCTGTTCGAAAAGATTA





GCAAC


 


150
NLpep75 (w/ Met)
A.A.
MNVTGWRLFEKISN


 


151
NLpep76 (w/ Met)
N.A.
ATGAACGTGAGCGGCTGGCGGCTGTTCGAAAAGATTA





GCAAC


 


152
NLpep76 (w/ Met)
A.A.
MNVSGWRLFEKISN


 


153
NLpep77 (w/ Met)
N.A.
ATG-





GTGACCGGCTGGCGGCTGTTCAAGAAGATTAGCAAC


 


154
NLpep77 (w/ Met)
A.A.
M-VTGWRLFKKISN


 


155
NLpep78 (w/ Met)
N.A.
ATGAACGTGAGCGGCTGGCGGCTGTTCAAGAAGATTA





GCAAC


 


156
NLpep78 (w/ Met)
A.A.
MNVSGWRLFKKISN


 


157
NLpep79 (w/ Met)
N.A.
ATGAACGTGACCGGCTACCGGCTGTTCAAGAAGATTA





GCAAC


 


158
NLpep79 (w/ Met)
A.A.
MNVTGYRLFKKISN


 


159
NLpep80 (w/ Met)
N.A.
ATGGTGAGCGGCTGGCGGCTGTTCAAGAAGATTAGCA





AC


 


160
NLpep80 (w/ Met)
A.A.
MVSGWRLFKKISN


 


161
NLpep81 (w/ Met)
N.A.
ATGAGCGGCTGGCGGCTGTTCAAGAAGATTAGCAAC


 


440
NLpep81 (w/ Met)
A.A.
MSGWRLFKKISN


 


163
NLpep82 (w/ Met)
N.A.
ATGGGCTGGCGGCTGTTCAAGAAGATTAGCAAC


 


164
NLpep82 (w/ Met)
A.A.
MGWRLFKKISN


 


165
NLpep83 (w/ Met)
N.A.
ATGAACGTGAGCGGCTGGCGGCTGTTCAAGAAGATTA





GC


 


166
NLpep83 (w/ Met)
A.A.
MNVSGWRLFKKIS


 


167
NLpep84 (w/ Met)
N.A.
ATGAACGTGAGCGGCTGGCGGCTGTTCAAGAAGATT


 


168
NLpep84 (w/ Met)
A.A.
MNVSGWRLFKKI


 


169
NLpep85 (w/ Met)
N.A.
ATGAACGTGAGCGGCTGGCGGCTGTTCAAGAAG


 


170
NLpep85 (w/ Met)
A.A.
MNVSGWRLFKK


 


171
NLpep86 (w/ Met)
N.A.
ATGGTGAGCGGCTGGCGGCTGTTCAAGAAGATTAGC


 


172
NLpep86 (w/ Met)
A.A.
MVSGWRLFKKIS


 


173
NLpep87 (w/ Met)
N.A.
ATGAGCGGCTGGCGGCTGTTCAAGAAGATT


 


174
NLpep87 (w/ Met)
A.A.
MSGWRLFKKI


 


175
NLpep88 (w/ Met)
N.A.
ATGAACGTGAGCGGCTGGGGCCTGTTCAAGAAGATTA





GCAAC


 


176
NLpep88 (w/ Met)
A.A.
MNVSGWGLFKKISN


 


177
NLpep89 (w/ Met)
N.A.
ATGCCCGTGAGCGGCTGGCGGCTGTTCAAGAAGATTA





GCAAC


 


178
NLpep89 (w/ Met)
A.A.
MPVSGWRLFKKISN


 


179
NLpep90 (w/ Met)
N.A.
ATGAACCCCGTGAGCGGCTGGCGGCTGTTCAAGAAGA





TTAGCAAC


 


180
NLpep90 (w/ Met)
A.A.
MNPVSGWRLFKKISN


 


181
NLpep91 (w/ Met)
N.A.
ATGATCAACCCCGTGAGCGGCTGGCGGCTGTTCAAGA





AGATTAGCAAC


 


182
NLpep91 (w/ Met)
A.A.
MINPVSGWRLFKKISN


 


183
NLpep92 (w/ Met)
N.A.
ATGACCATCAACCCCGTGAGCGGCTGGCGGCTGTTCAA





GAAGATTAGCAAC


 


184
NLpep92 (w/ Met)
A.A.
MTINPVSGWRLFKKISN


 


185
NLpep93 (w/ Met)
N.A.
ATGGTGACCATCAACCCCGTGAGCGGCTGGCGGCTGTT





CAAGAAGATTAGCAAC


 


186
NLpep93 (w/ Met)
A.A.
MVTINPVSGWRLFKKISN


 


187
NLpep94 (w/ Met)
N.A.
ATGCGGGTGACCATCAACCCCGTGAGCGGCTGGCGGC





TGTTCAAGAAGATTAGCAAC


 


188
NLpep94 (w/ Met)
A.A.
MRVTINPVSGWRLFKKISN


 


189
NLpep95 (w/ Met)
N.A.
ATGAGCGGCTGGCGGCTGCTGAAGAAGATT


 


190
NLpep95 (w/ Met)
A.A.
MSGWRLLKKI


 


191
NLpep96 (w/ Met)
N.A.
ATGACCGGCTACCGGCTGCTGAAGAAGATT


 


192
NLpep96 (w/ Met)
A.A.
MTGYRLLKKI


 


193
NLpep97 (w/ Met)
N.A.
ATGAGCGGCTGGCGGCTGTTCAAGAAG


 


194
NLpep97 (w/ Met)
A.A.
MSGWRLFKK


 


195
NLpep98 (w/ Met)
N.A.
ATGGTGACCGGCTACCGGCTGTTCAAGAAGATTAGC


 


196
NLpep98 (w/ Met)
A.A.
MVTGYRLFKKIS


 


197
NLpep99 (w/ Met)
N.A.
ATGGTGACCGGCTACCGGCTGTTCGAGAAGATTAGC


 


198
NLpep99 (w/ Met)
A.A.
MVTGYRLFEKIS


 


199
NLpep100 (w/ Met)
N.A.
ATGGTGACCGGCTACCGGCTGTTCGAGCAGATTAGC


 


200
NLpep100 (w/ Met)
A.A.
MVTGYRLFEQIS


 


201
NLpep101 (w/ Met)
N.A.
ATGGTGACCGGCTACCGGCTGTTCGAGAAGGAGAGC


 


202
NLpep101 (w/ Met)
A.A.
MVTGYRLFEKES


 


203
NLpep102 (w/ Met)
N.A.
ATGGTGACCGGCTACCGGCTGTTCGAGCAGGAGAGC


 


204
NLpep102 (w/ Met)
A.A.
MVTGYRLFEQES


 


205
NLpep103 (w/ Met)
N.A.
ATGGTGACCGGCTACCGGCTGTTCGAGCAGGAGCTG


 


206
NLpep103 (w/ Met)
A.A.
MVTGYRLFEQEL


 


207
NLpep104 (w/ Met)
N.A.
ATGGTGGAGGGCTACCGGCTGTTCGAGAAGATTAGC


 


208
NLpep104 (w/ Met)
A.A.
MVEGYRLFEKIS


 


209
NLpep105 (w/ Met)
N.A.
ATGGTGGAGGGCTACCGGCTGTTCGAGCAGATTAGC


 


210
NLpep105 (w/ Met)
A.A.
MVEGYRLFEQIS


 


211
NLpep106 (w/ Met)
N.A.
ATGGTGGAGGGCTACCGGCTGTTCGAGAAGGAGAGC


 


212
NLpep106 (w/ Met)
A.A.
MVEGYRLFEKES


 


213
NLpep107 (w/ Met)
N.A.
ATGGTGGAGGGCTACCGGCTGTTCGAGCAGGAGAGC


 


214
NLpep107 (w/ Met)
A.A.
MVEGYRLFEQES


 


215
NLpep108 (w/ Met)
N.A.
ATGGTGGAGGGCTACCGGCTGTTCGAGCAGGAGCTG


 


216
NLpep108 (w/ Met)
A.A.
MVEGYRLFEQEL


 


217
NLpep109 (w/ Met)
N.A.
ATGATTAGCGGCTGGCGGCTGATGAAGAACATTAGC


 


218
NLpep109 (w/ Met)
A.A.
MISGWRLMKNIS


 


219
NLpep110 (w/ Met)
N.A.
ATGGTGGAGGGCTACCGGCTGTTCAAGAAGATTAGC


 


220
NLpep110 (w/ Met)
A.A.
MVEGYRLFKKIS


 


221
NLpep2 (w/o Met)
N.A.
GACGTGACCGGCTGGCGGCTGTGCGAACGCATTCTGG





CG


 


222
NLpep2 (w/o Met)
A.A.
DVTGWRLCERILA


 


223
NLpep3 (w/o Met)
N.A.
GGAGTGACCGCCTGGCGGCTGTGCGAACGCATTCTGG





CG


 


224
NLpep3 (w/o Met)
A.A.
GVTAWRLCERILA


 


225
NLpep4 (w/o Met)
N.A.
GGAGTGACCGGCTGGCGGCTGTGCAAGCGCATTCTGG





CG


 


226
NLpep4 (w/o Met)
A.A.
GVTGWRLCKRILA


 


227
NLpep5 (w/o Met)
N.A.
GGAGTGACCGGCTGGCGGCTGTGCGAACGCATTAGCG





CG


 


228
NLpep5 (w/o Met)
A.A.
GVTGWRLCERISA


 


229
NLpep6 (w/o Met)
N.A.
GACGTGACCGGCTGGCGGCTGTGCAAGCGCATTAGCG





CG


 


230
NLpep6 (w/o Met)
A.A.
DVTGWRLCKRISA


 


231
NLpep7 (w/o Met)
N.A.
GACGTGACCGGCTGGCGGCTGTGCAAGCGCATTCTGG





CG


 


232
NLpep7 (w/o Met)
A.A.
DVTGWRLCKRILA


 


233
NLpep8 (w/o Met)
N.A.
GACGTGACCGGCTGGCGGCTGTGCGAACGCATTAGCG





CG


 


234
NLpep8 (w/o Met)
A.A.
DVTGWRLCERISA


 


235
NLpep9 (w/o Met)
N.A.
GGAGTGACCGGCTGGCGGCTGTGCAAGCGCATTAGCG





CG


 


236
NLpep9 (w/o Met)
A.A.
GVTGWRLCKRISA


 


237
NLpep10 (w/o Met)
N.A.
GGAGTGACCGGCTGGCGGCTGAACGAACGCATTCTGG





CG


 


238
NLpep10 (w/o Met)
A.A.
GVTGWRLNERILA


 


239
NLpep11 (w/o Met)
N.A.
GGAGTGACCGGCTGGCGGCTGCAGGAACGCATTCTGG





CG


 


240
NLpep11 (w/o Met)
A.A.
GVTGWRLQERILA


 


241
NLpep12 (w/o Met)
N.A.
GGAGTGACCGGCTGGCGGCTGAAGAAGCGCCGGAGCC





GG


 


242
NLpep12 (w/o Met)
A.A.
GVTGWRLKKRRSR


 


243
NLpep13 (w/o Met)
N.A.
AACGTGACCGGCTGGCGGCTGTGCAAGCGCATTAGCG





CG


 


244
NLpep13 (w/o Met)
A.A.
NVTGWRLCKRISA


 


245
NLpep14 (w/o Met)
N.A.
AGCGTGACCGGCTGGCGGCTGTGCAAGCGCATTAGCG





CG


 


246
NLpep14 (w/o Met)
A.A.
SVTGWRLCKRISA


 


247
NLpep15 (w/o Met)
N.A.
GAGGTGACCGGCTGGCGGCTGTGCAAGCGCATTAGCG





CG


 


248
NLpep15 (w/o Met)
A.A.
EVTGWRLCKRISA


 


249
NLpep16 (w/o Met)
N.A.
GGCGTGACCGGCTGGCGGCTGTGCAAGCGCATTAGCG





CG


 


250
NLpep16 (w/o Met)
A.A.
HVTGWRLCKRISA


 


251
NLpep17 (w/o Met)
N.A.
GGACACACCGGCTGGCGGCTGTGCAAGCGCATTAGCG





CG


 


252
NLpep17 (w/o Met)
A.A.
GITGWRLCKRISA


 


253
NLpep18 (w/o Met)
N.A.
GGAGCCACCGGCTGGCGGCTGTGCAAGCGCATTAGCG





CG


 


254
NLpep18 (w/o Met)
A.A.
GATGWRLCKRISA


 


255
NLpep19 (w/o Met)
N.A.
GGAAAGACCGGCTGGCGGCTGTGCAAGCGCATTAGCG





CG


 


256
NLpep19 (w/o Met)
A.A.
GKTGWRLCKRISA


 


257
NLpep20 (w/o Met)
N.A.
GGACAGACCGGCTGGCGGCTGTGCAAGCGCATTAGCG





CG


 


258
NLpep20 (w/o Met)
A.A.
GQTGWRLCKRISA


 


259
NLpep21 (w/o Met)
N.A.
GGAAGCACCGGCTGGCGGCTGTGCAAGCGCATTAGCG





CG


 


260
NLpep21 (w/o Met)
A.A.
GSTGWRLCKRISA


 


261
NLpep22 (w/o Met)
N.A.
GGAGTGGTGGGCTGGCGGCTGTGCAAGCGCATTAGCG





CG


 


262
NLpep22 (w/o Met)
A.A.
GVVGWRLCKRISA


 


263
NLpep23 (w/o Met)
N.A.
GGAGTGAAGGGCTGGCGGCTGTGCAAGCGCATTAGCG





CG


 


264
NLpep23 (w/o Met)
A.A.
GVKGWRLCKRISA


 


265
NLpep24 (w/o Met)
N.A.
GGAGTGCAGGGCTGGCGGCTGTGCAAGCGCATTAGCG





CG


 


266
NLpep24 (w/o Met)
A.A.
GVQGWRLCKRISA


 


267
NLpep25 (w/o Met)
N.A.
GGAGTGACCGGCACCCGGCTGTGCAAGCGCATTAGCG





CG


 


268
NLpep25 (w/o Met)
A.A.
GVTGTRLCKRISA


 


269
NLpep26 (w/o Met)
N.A.
GGAGTGACCGGCAAGCGGCTGTGCAAGCGCATTAGCG





CG


 


270
NLpep26 (w/o Met)
A.A.
GVTGKRLCKRISA


 


271
NLpep27 (w/o Met)
N.A.
GGAGTGACCGGCGTGCGGCTGTGCAAGCGCATTAGCG





CG


 


272
NLpep27 (w/o Met)
A.A.
GVTGVRLCKRISA


 


273
NLpep28 (w/o Met)
N.A.
GGAGTGACCGGCTGGCGGCACTGCAAGCGCATTAGCG





CG


 


274
NLpep28 (w/o Met)
A.A.
GVTGWRICKRISA


 


275
NLpep29 (w/o Met)
N.A.
GGAGTGACCGGCTGGCGGGTGTGCAAGCGCATTAGCG





CG


 


276
NLpep29 (w/o Met)
A.A.
GVTGWRVCKRISA


 


277
NLpep30 (w/o Met)
N.A.
GGAGTGACCGGCTGGCGGACCTGCAAGCGCATTAGCG





CG


 


278
NLpep30 (w/o Met)
A.A.
GVTGWRTCKRISA


 


279
NLpep31 (w/o Met)
N.A.
GGAGTGACCGGCTGGCGGTACTGCAAGCGCATTAGCG





CG


 


280
NLpep31 (w/o Met)
A.A.
GVTGWRYCKRISA


 


281
NLpep32 (w/o Met)
N.A.
GGAGTGACCGGCTGGCGGAAGTGCAAGCGCATTAGCG





CG


 


282
NLpep32 (w/o Met)
A.A.
GVTGWRKCKRISA


 


283
NLpep33 (w/o Met)
N.A.
GGAGTGACCGGCTGGCGGCTGAACAAGCGCATTAGCG





CG


 


284
NLpep33 (w/o Met)
A.A.
GVTGWRLNKRISA


 


285
NLpep34 (w/o Met)
N.A.
GGAGTGACCGGCTGGCGGCTGACCAAGCGCATTAGCG





CG


 


286
NLpep34 (w/o Met)
A.A.
GVTGWRLTKRISA


 


287
NLpep35 (w/o Met)
N.A.
GGAGTGACCGGCTGGCGGCTGTGCAAGAAGATTAGCG





CG


 


288
NLpep35 (w/o Met)
A.A.
GVTGWRLCKKISA


 


289
NLpep36 (w/o Met)
N.A.
GGAGTGACCGGCTGGCGGCTGTGCAAGAACATTAGCG





CG


 


290
NLpep36 (w/o Met)
A.A.
GVTGWRLCKNISA


 


291
NLpep37 (w/o Met)
N.A.
GGAGTGACCGGCTGGCGGCTGTGCAAGCGCGTGAGCG





CG


 


292
NLpep37 (w/o Met)
A.A.
GVTGWRLCKRVSA


 


293
NLpep38 (w/o Met)
N.A.
GGAGTGACCGGCTGGCGGCTGTGCAAGCGCCAGAGCG





CG


 


294
NLpep38 (w/o Met)
A.A.
GVTGWRLCKRQSA


 


295
NLpep39 (w/o Met)
N.A.
GGAGTGACCGGCTGGCGGCTGTGCAAGCGCGAGAGCG





CG


 


296
NLpep39 (w/o Met)
A.A.
GVTGWRLCKRESA


 


297
NLpep40 (w/o Met)
N.A.
GGAGTGACCGGCTGGCGGCTGTGCAAGCGCCGGAGCG





CG


 


298
NLpep40 (w/o Met)
A.A.
GVTGWRLCKRRSA


 


299
NLpep41 (w/o Met)
N.A.
GGAGTGACCGGCTGGCGGCTGTGCAAGCGCTTCAGCG





CG


 


300
NLpep41 (w/o Met)
A.A.
GVTGWRLCKRFSA


 


301
NLpep42 (w/o Met)
N.A.
GGAGTGACCGGCTGGCGGCTGTGCAAGCGCATTAGCA





AC


 


302
NLpep42 (w/o Met)
A.A.
GVTGWRLCKRISN


 


303
NLpep43 (w/o Met)
N.A.
GGAGTGACCGGCTGGCGGCTGTGCAAGCGCATTAGCA





CC


 


304
NLpep43 (w/o Met)
A.A.
GVTGWRLCKRIST


 


305
NLpep44 (w/o Met)
N.A.
GGAGTGACCGGCTGGCGGCTGTGCAAGCGCATTAGCC





GG


 


306
NLpep44 (w/o Met)
A.A.
GVTGWRLCKRISR


 


307
NLpep45 (w/o Met)
N.A.
GGAGTGACCGGCTGGCGGCTGTGCAAGCGCATTAGCC





TG


 


308
NLpep45 (w/o Met)
A.A.
GVTGWRLCKRISL


 


309
NLpep46 (w/o Met)
N.A.
GGAGTGACCGGCTGGCGGCTGTGCAAGCGCATTAGCG





AG


 


310
NLpep46 (w/o Met)
A.A.
GVTGWRLCKRISE


 


311
NLpep47 (w/o Met)
N.A.
GGAGTGACCGGCTTCCGGCTGTGCAAGCGCATTAGCG





CG


 


312
NLpep47 (w/o Met)
A.A.
GVTGFRLCKRISA


 


313
NLpep48 (w/o Met)
N.A.
GGAGTGACCGGCTACCGGCTGTGCAAGCGCATTAGCG





CG


 


314
NLpep48 (w/o Met)
A.A.
GVTGYRLCKRISA


 


315
NLpep49 (w/o Met)
N.A.
GGAGTGACCGGCGAGCGGCTGTGCAAGCGCATTAGCG





CG


 


316
NLpep49 (w/o Met)
A.A.
GVTGERLCKRISA


 


317
NLpep50 (w/o Met)
N.A.
CAGGTGACCGGCTGGCGGCTGTGCAAGCGCATTAGCG





CG


 


318
NLpep50 (w/o Met)
A.A.
QVTGWRLCKRISA


 


319
NLpep51 (w/o Met)
N.A.
ACCGTGACCGGCTGGCGGCTGTGCAAGCGCATTAGCG





CG


 


320
NLpep51 (w/o Met)
A.A.
TVTGWRLCKRISA


 


321
NLpep52 (w/o Met)
N.A.
GGAGTGGAGGGCTGGCGGCTGTGCAAGCGCATTAGCG





CG


 


322
NLpep52 (w/o Met)
A.A.
GVEGWRLCKRISA


 


323
NLpep53 (w/o Met)
N.A.
GGAGTGACCGGCTGGCGGCTGTTCAAGCGCATTAGCG





CG


 


324
NLpep53 (w/o Met)
A.A.
GVTGWRLFKRISA


 


325
NLpep54 (w/o Met)
N.A.
GGAGTGACCGGCTGGCGGCTGTACAAGCGCATTAGCG





CG


 


326
NLpep54 (w/o Met)
A.A.
GVTGWRLYKRISA


 


327
NLpep55 (w/o Met)
N.A.
GGAGTGACCGGCTGGCGGCTGAGCAAGCGCATTAGCG





CG


 


328
NLpep55 (w/o Met)
A.A.
GVTGWRLSKRISA


 


329
NLpep56 (w/o Met)
N.A.
GGAGTGACCGGCTGGCGGCTGGGCAAGCGCATTAGCG





CG


 


330
NLpep56 (w/o Met)
A.A.
GVTGWRLHKRISA


 


331
NLpep57 (w/o Met)
N.A.
GGAGTGACCGGCTGGCGGCTGATGAAGCGCATTAGCG





CG


 


332
NLpep57 (w/o Met)
A.A.
GVTGWRLMKRISA


 


333
NLpep58 (w/o Met)
N.A.
GGAGTGACCGGCTGGCGGCTGGCCAAGCGCATTAGCG





CG


 


334
NLpep58 (w/o Met)
A.A.
GVTGWRLAKRISA


 


335
NLpep59 (w/o Met)
N.A.
GGAGTGACCGGCTGGCGGCTGCAGAAGCGCATTAGCG





CG


 


336
NLpep59 (w/o Met)
A.A.
GVTGWRLQKRISA


 


337
NLpep60 (w/o Met)
N.A.
GGAGTGACCGGCTGGCGGCTGCTGAAGCGCATTAGCG





CG


 


338
NLpep60 (w/o Met)
A.A.
GVTGWRLLKRISA


 


339
NLpep61 (w/o Met)
N.A.
GGAGTGACCGGCTGGCGGCTGAAGAAGCGCATTAGCG





CG


 


340
NLpep61 (w/o Met)
A.A.
GVTGWRLKKRISA


 


341
NLpep62 (w/o Met)
N.A.
AACCACACCGGCTGGCGGCTGAACAAGAAGGTGAGCA





AC


 


342
NLpep62 (w/o Met)
A.A.
NITGWRLNKKVSN


 


343
NLpep63 (w/o Met)
N.A.
AACCACACCGGCTACCGGCTGAACAAGAAGGTGAGCA





AC


 


344
NLpep63 (w/o Met)
A.A.
NITGYRLNKKVSN


 


345
NLpep64 (w/o Met)
N.A.
TGCGTGACCGGCTGGCGGCTGTTCAAGCGCATTAGCGC





G


 


346
NLpep64 (w/o Met)
A.A.
CVTGWRLFKRISA


 


347
NLpep65 (w/o Met)
N.A.
CCCGTGACCGGCTGGCGGCTGTTCAAGCGCATTAGCGC





G


 


348
NLpep65 (w/o Met)
A.A.
PVTGWRLFKRISA


 


349
NLpep66 (w/o Met)
N.A.
AACCACACCGGCTACCGGCTGTTCAAGAAGGTGAGCA





AC


 


350
NLpep66 (w/o Met)
A.A.
NITGYRLFKKVSN


 


351
NLpep67 (w/o Met)
N.A.
AACGTGACCGGCTACCGGCTGTTCAAGAAGGTGAGCA





AC


 


352
NLpep67 (w/o Met)
A.A.
NVTGYRLFKKVSN


 


353
NLpep68 (w/o Met)
N.A.
AACGTGACCGGCTGGCGGCTGTTCAAGAAGGTGAGCA





AC


 


354
NLpep68 (w/o Met)
A.A.
NVTGWRLFKKVSN


 


355
NLpep69 (w/o Met)
N.A.
AACGTGACCGGCTGGCGGCTGTTCAAGAAGATTAGCA





AC


 


356
NLpep69 (w/o Met)
A.A.
NVTGWRLFKKISN


 


357
NLpep70 (w/o Met)
N.A.
AACGTGACCGGCTGGCGGCTGTTCAAGCGCATTAGCA





AC


 


358
NLpep70 (w/o Met)
A.A.
NVTGWRLFKRISN


 


359
NLpep71 (w/o Met)
N.A.
GGAGTGACCGGCTGGCGGCTGTTCAAGCGCATTAGCA





AC


 


360
NLpep71 (w/o Met)
A.A.
GVTGWRLFKRISN


 


361
NLpep72 (w/o Met)
N.A.
AACGTGACCGGCTGGCGGCTGTTCGAACGCATTAGCA





AC


 


362
NLpep72 (w/o Met)
A.A.
NVTGWRLFERISN


 


363
NLpep73 (w/o Met)
N.A.
AACGTGACCGGCTGGCGGCTGTTCAAGCGCATTCTGAA





C


 


364
NLpep73 (w/o Met)
A.A.
NVTGWRLFKRILN


 


365
NLpep74 (w/o Met)
N.A.
AACGTGACCGGCTGGCGGCTGTTCAAGCGCATTAGCG





CG


 


366
NLpep74 (w/o Met)
A.A.
NVTGWRLFKRISA


 


367
NLpep75 (w/o Met)
N.A.
AACGTGACCGGCTGGCGGCTGTTCGAAAAGATTAGCA





AC


 


368
NLpep75 (w/o Met)
A.A.
NVTGWRLFEKISN


 


369
NLpep76 (w/o Met)
N.A.
AACGTGAGCGGCTGGCGGCTGTTCGAAAAGATTAGCA





AC


 


370
NLpep76 (w/o Met)
A.A.
NVSGWRLFEKISN


 


371
NLpep77 (w/o Met)
N.A.
GTGACCGGCTGGCGGCTGTTCAAGAAGATTAGCAAC


 


372
NLpep77 (w/o Met)
A.A.
VTGWRLFKKISN


 


373
NLpep78 (w/o Met)
N.A.
AACGTGAGCGGCTGGCGGCTGTTCAAGAAGATTAGCA





AC


 


374
NLpep78 (w/o Met)
A.A.
NVSGWRLFKKISN


 


375
NLpep79 (w/o Met)
N.A.
AACGTGACCGGCTACCGGCTGTTCAAGAAGATTAGCA





AC


 


376
NLpep79 (w/o Met)
A.A.
NVTGYRLFKKISN


 


377
NLpep80 (w/o Met)
N.A.
GTGAGCGGCTGGCGGCTGTTCAAGAAGATTAGCAAC


 


378
NLpep80 (w/o Met)
A.A.
VSGWRLFKKISN


 


379
NLpep81 (w/o Met)
N.A.
AGCGGCTGGCGGCTGTTCAAGAAGATTAGCAAC


 


380
NLpep81 (w/o Met)
A.A.
SGWRLFKKISN


 


381
NLpep82 (w/o Met)
N.A.
GGCTGGCGGCTGTTCAAGAAGATTAGCAAC


 


382
NLpep82 (w/o Met)
A.A.
GWRLFKKISN


 


383
NLpep83 (w/o Met)
N.A.
AACGTGAGCGGCTGGCGGCTGTTCAAGAAGATTAGC


 


384
NLpep83 (w/o Met)
A.A.
NVSGWRLFKKIS


 


385
NLpep84 (w/o Met)
N.A.
AACGTGAGCGGCTGGCGGCTGTTCAAGAAGATT


 


386
NLpep84 (w/o Met)
A.A.
NVSGWRLFKKI


 


387
NLpep85 (w/o Met)
N.A.
AACGTGAGCGGCTGGCGGCTGTTCAAGAAG


 


388
NLpep85 (w/o Met)
A.A.
NVSGWRLFKK


 


389
NLpep86 (w/o Met)
N.A.
GTGAGCGGCTGGCGGCTGTTCAAGAAGATTAGC


 


390
NLpep86 (w/o Met)
A.A.
VSGWRLFKKIS


 


391
NLpep87 (w/o Met)
N.A.
AGCGGCTGGCGGCTGTTCAAGAAGATT


 


392
NLpep87 (w/o Met)
A.A.
SGWRLFKKI


 


393
NLpep88 (w/o Met)
N.A.
AACGTGAGCGGCTGGGGCCTGTTCAAGAAGATTAGCA





AC


 


394
NLpep88 (w/o Met)
A.A.
NVSGWGLFKKISN


 


395
NLpep89 (w/o Met)
N.A.
CCCGTGAGCGGCTGGCGGCTGTTCAAGAAGATTAGCA





AC


 


396
NLpep89 (w/o Met)
A.A.
PVSGWRLFKKISN


 


397
NLpep90 (w/o Met)
N.A.
AACCCCGTGAGCGGCTGGCGGCTGTTCAAGAAGATTA





GCAAC


 


398
NLpep90 (w/o Met)
A.A.
NPVSGWRLFKKISN


 


399
NLpep91 (w/o Met)
N.A.
ATCAACCCCGTGAGCGGCTGGCGGCTGTTCAAGAAGA





TTAGCAAC


 


400
NLpep91 (w/o Met)
A.A.
INPVSGWRLFKKISN


 


401
NLpep92 (w/o Met)
N.A.
ACCATCAACCCCGTGAGCGGCTGGCGGCTGTTCAAGA





AGATTAGCAAC


 


402
NLpep92 (w/o Met)
A.A.
TINPVSGWRLFKKISN


 


403
NLpep93 (w/o Met)
N.A.
GTGACCATCAACCCCGTGAGCGGCTGGCGGCTGTTCAA





GAAGATTAGCAAC


 


404
NLpep93 (w/o Met)
A.A.
VTINPVSGWRLFKKISN


 


405
NLpep94 (w/o Met)
N.A.
CGGGTGACCATCAACCCCGTGAGCGGCTGGCGGCTGTT





CAAGAAGATTAGCAAC


 


406
NLpep94 (w/o Met)
A.A.
RVTINPVSGWRLFKKISN


 


407
NLpep95 (w/o Met)
N.A.
AGCGGCTGGCGGCTGCTGAAGAAGATT


 


408
NLpep95 (w/o Met)
A.A.
SGWRLLKKI


 


409
NLpep96 (w/o Met)
N.A.
ACCGGCTACCGGCTGCTGAAGAAGATT


 


410
NLpep96 (w/o Met)
A.A.
TGYRLLKKI


 


411
NLpep97 (w/o Met)
N.A.
AGCGGCTGGCGGCTGTTCAAGAAG


 


412
NLpep97 (w/o Met)
A.A.
SGWRLFKK


 


413
NLpep98 (w/o Met)
N.A.
GTGACCGGCTACCGGCTGTTCAAGAAGATTAGC


 


414
NLpep98 (w/o Met)
A.A.
VTGYRLFKKIS


 


415
NLpep99 (w/o Met)
N.A.
GTGACCGGCTACCGGCTGTTCGAGAAGATTAGC


 


416
NLpep99 (w/o Met)
A.A.
VTGYRLFKKIS


 


417
NLpep100 (w/o Met)
N.A.
GTGACCGGCTACCGGCTGTTCGAGCAGATTAGC


 


418
NLpep100 (w/o Met)
A.A.
VTGYRLFEQIS


 


419
NLpep101 (w/o Met)
N.A.
GTGACCGGCTACCGGCTGTTCGAGAAGGAGAGC


 


420
NLpep101 (w/o Met)
A.A.
VTGYRLFEKES


 


421
NLpep102 (w/o Met)
N.A.
GTGACCGGCTACCGGCTGTTCGAGCAGGAGAGC


 


422
NLpep102 (w/o Met)
A.A.
VTGYRLFEQES


 


423
NLpep103 (w/o Met)
N.A.
GTGACCGGCTACCGGCTGTTCGAGCAGGAGCTG


 


424
NLpep103 (w/o Met)
A.A.
VTGYRLFEQEL


 


425
NLpep104 (w/o Met)
N.A.
GTGGAGGGCTACCGGCTGTTCGAGAAGATTAGC


 


426
NLpep104 (w/o Met)
A.A.
VEGYRLFEKIS


 


427
NLpep105 (w/o Met)
N.A.
GTGGAGGGCTACCGGCTGTTCGAGCAGATTAGC


 


428
NLpep105 (w/o Met)
A.A.
VEGYRLFEQIS


 


429
NLpep106 (w/o Met)
N.A.
GTGGAGGGCTACCGGCTGTTCGAGAAGGAGAGC


 


430
NLpep106 (w/o Met)
A.A.
VEGYRLFEKES


 


431
NLpep107 (w/o Met)
N.A.
GTGGAGGGCTACCGGCTGTTCGAGCAGGAGAGC


 


432
NLpep107 (w/o Met)
A.A.
VEGYRLFEQES


 


433
NLpep108 (w/o Met)
N.A.
GTGGAGGGCTACCGGCTGTTCGAGCAGGAGCTG


 


434
NLpep108 (w/o Met)
A.A.
VEGYRLFEQEL


 


435
NLpep109 (w/o Met)
N.A.
ATTAGCGGCTGGCGGCTGATGAAGAACATTAGC


 


436
NLpep109 (w/o Met)
A.A.
ISGWRLMKNIS


 


437
NLpep110 (w/o Met)
N.A.
GTGGAGGGCTACCGGCTGTTCAAGAAGATTAGC


 


438
NLpep110 (w/o Met)
A.A.
VEGYRLFKKIS


 


2162
NLpep111 (w/ Met)
N.A.
ATGGTGACCGGCTACCGGCTGTTCGAGGAGATCAGC


 


2163
NLpep111 (w/ Met)
A.A.
MVTGYRLFEEIS


 


2164
NLpep112 (w/ Met)
N.A.
ATGGTGACCGGCTACCGGCTGTTCGAGGAGGCCAGC


 


2165
NLpep112 (w/ Met)
A.A.
MVTGYRLFEEAS


 


2166
NLpep113 (w/ Met)
N.A.
ATGGTGACCGGCTACCGGCTGTTCGAGGAGGAGAGC


 


2167
NLpep113 (w/ Met)
A.A.
MVTGYRLFEEES


 


2168
NLpep114 (w/ Met)
N.A.
ATGGTGACCGGCTACCGGCTGTTCGAGGAGATCCTG


 


2169
NLpep114 (w/ Met)
A.A.
MVTGYRLFEEIL


 


2170
NLpep115 (w/ Met)
N.A.
ATGGTGACCGGCTACCGGCTGTTCGAGGAGGCCCTG


 


2171
NLpep115 (w/ Met)
A.A.
MVTGYRLFEEAL


 


2172
NLpep116 (w/ Met)
N.A.
ATGGTGACCGGCTACCGGCTGTTCGAGGAGGAGCTG


 


2173
NLpep116 (w/ Met)
A.A.
MVTGYRLFEEEL


 


2174
NLpep117 (w/ Met)
N.A.
ATGGTGGAGGGCTACCGGCTGTTCGAGGAGATCAGC


 


2175
NLpep117 (w/ Met)
A.A.
MVEGYRLFEEIS


 


2176
NLpep118 (w/ Met)
N.A.
ATGGTGGAGGGCTACCGGCTGTTCGAGGAGGCCAGC


 


2177
NLpep118 (w/ Met)
A.A.
MVEGYRLFEEAS


 


2178
NLpep119 (w/ Met)
N.A.
ATGGTGGAGGGCTACCGGCTGTTCGAGGAGGAGAGC


 


2179
NLpep119 (w/ Met)
A.A.
MVEGYRLFEEES


 


2180
NLpep120 (w/ Met)
N.A.
ATGGTGGAGGGCTACCGGCTGTTCGAGGAGATCCTG


 


2181
NLpep120 (w/ Met)
A.A.
MVEGYRLFEEIL


 


2182
NLpep121 (w/ Met)
N.A.
ATGGTGGAGGGCTACCGGCTGTTCGAGGAGGCCCTG


 


2183
NLpep121 (w/ Met)
A.A.
MVEGYRLFEEAL


 


2184
NLpep122 (w/ Met)
N.A.
ATGGTGGAGGGCTACCGGCTGTTCGAGGAGGAGCTG


 


2185
NLpep122 (w/ Met)
A.A.
MVEGYRLFEEEL


 


2186
NLpep123 (w/ Met)
N.A.
ATGGTGACCGGCTACCGGCTGTTCAAGAAGATCCTG


 


2187
NLpep123 (w/ Met)
A.A.
MVTGYRLFKKIL


 


2188
NLpep124 (w/ Met)
N.A.
ATGGTGACCGGCTACCGGCTGATGAAGAAGATCCTG


 


2189
NLpep124 (w/ Met)
A.A.
MVTGYRLMKKIL


 


2190
NLpep125 (w/ Met)
N.A.
ATGGTGACCGGCTACCGGCTGCACAAGAAGATCCTG


 


2191
NLpep125 (w/ Met)
A.A.
MVTGYRLHKKIL


 


2192
NLpep126 (w/ Met)
N.A.
ATGGTGACCGGCTACCGGCTGCTGAAGAAGATCCTG


 


2193
NLpep126 (w/ Met)
A.A.
MVTGYRLLKKIL


 


2194
NLpep127 (w/ Met)
N.A.
ATGGTGACCGGCTACCGGCTGAGCAAGAAGATCCTG


 


2195
NLpep127 (w/ Met)
A.A.
MVTGYRLSKKIL


 


2196
NLpep128 (w/ Met)
N.A.
ATGGTGACCGGCTACCGGCTGTTCGAGAAGATCCTG


 


2197
NLpep128 (w/ Met)
A.A.
MVTGYRLFEKIL


 


2198
NLpep129 (w/ Met)
N.A.
ATGGTGACCGGCTACCGGCTGATGGAGAAGATCCTG


 


2199
NLpep129 (w/ Met)
A.A.
MVTGYRLMEKIL


 


2200
NLpep130 (w/ Met)
N.A.
ATGGTGACCGGCTACCGGCTGCACGAGAAGATCCTG


 


2201
NLpep130 (w/ Met)
A.A.
MVTGYRLHEKIL


 


2202
NLpep131 (w/ Met)
N.A.
ATGGTGACCGGCTACCGGCTGCTGGAGAAGATCCTG


 


2203
NLpep131 (w/ Met)
A.A.
MVTGYRLLEKIL


 


2204
NLpep132 (w/ Met)
N.A.
ATGGTGACCGGCTACCGGCTGAGCGAGAAGATCCTG


 


2205
NLpep132 (w/ Met)
A.A.
MVTGYRLSEKIL


 


2206
NLpep133 (w/ Met)
N.A.
ATGGTGACCGGCTACCGGCTGATGGAGGAGATCCTG


 


2207
NLpep133 (w/ Met)
A.A.
MVTGYRLMEEIL


 


2208
NLpep134 (w/ Met)
N.A.
ATGGTGACCGGCTACCGGCTGCACGAGGAGATCCTG


 


2209
NLpep134 (w/ Met)
A.A.
MVTGYRLHEEIL


 


2210
NLpep135 (w/ Met)
N.A.
ATGGTGACCGGCTACCGGCTGCTGGAGGAGATCCTG


 


2211
NLpep135 (w/ Met)
A.A.
MVTGYRLLEEIL


 


2212
NLpep136 (w/ Met)
N.A.
ATGGTGACCGGCTACCGGCTGAGCGAGGAGATCCTG


 


2213
NLpep136 (w/ Met)
A.A.
MVTGYRLSEEIL


 


2214
NLpep137 (w/ Met)
N.A.
ATGGTGAGCGGCTACCGGCTGTTCGAGGAGATCCTG


 


2215
NLpep137 (w/ Met)
A.A.
MVSGYRLFEEIL


 


2216
NLpep138 (w/ Met)
N.A.
ATGGTGACCGGCTGGCGGCTGTTCGAGGAGATCCTG


 


2217
NLpep138 (w/ Met)
A.A.
MVTGWRLFEEIL


 


2218
NLpep139 (w/ Met)
N.A.
ATGGTGAGCGGCTGGCGGCTGTTCGAGGAGATCCTG


 


2219
NLpep139 (w/ Met)
A.A.
MVSGWRLFEEIL


 


2220
NLpep140 (w/ Met)
N.A.
ATGAACGTGACCGGCTACCGGCTGTTCGAGGAGATC





CTG


 


2221
NLpep140 (w/ Met)
A.A.
MNVTGYRLFEEIL


 


2222
NLpep141 (w/ Met)
N.A.
ATGGTGACCGGCTACCGGCTGTTCGAGGAGATCCTG





AAC


 


2223
NLpep141 (w/ Met)
A.A.
MVTGYRLFEEILN


 


2224
NLpep142 (w/ Met)
N.A.
ATGAACGTGACCGGCTACCGGCTGTTCGAGGAGATC





CTGAAC


 


2225
NLpep142 (w/ Met)
A.A.
MNVTGYRLFEEILN


 


2226
NLpep143 (w/ Met)
N.A.
ATGGTGACCGGCTACCGGCTGTTCGAGGAGATC


 


2227
NLpep143 (w/ Met)
A.A.
MVTGYRLFEEI


 


2228
NLpep144 (w/ Met)
N.A.
ATGGTGACCGGCTACCGGCTGTTCCAGAAGATCAGC


 


2229
NLpep144 (w/ Met)
A.A.
MVTGYRLFQKIS


 


2230
NLpep145 (w/ Met)
N.A.
ATGGTGACCGGCTACCGGCTGTTCAAGAAGATCAGCA





AC


 


2231
NLpep145 (w/ Met)
A.A.
MVTGYRLFKKISN


 


2232
NLpep146 (w/ Met)
N.A.
ATGGTGACCGGCTACCGGCTGTTCAAGAAGATCAGC


 


2233
NLpep146 (w/ Met)
A.A.
MVTGYRLFKKIS


 


2234
NLpep147 (w/ Met)
A.A.
MVSGWRLFKKISA


 


2235
NLpep148 (w/ Met)
A.A.
MGVSGWRLFKKIS


 


2236
NLpep149 (w/ Met)
A.A.
MSVSGWRLFKKISN


 


2237
NLpep150 (w/ Met)
A.A.
MSVSGWRLFKKISA


 


2238
NLpep151 (w/ Met)
A.A.
MNSVSGWRLFKKISA


 


2239
NLpep152 (w/ Met)
A.A.
MNSVSGWRLFKKISN


 


2240
NLpep153 (w/ Met)
A.A.
MSNVSGWRLFKKIS


 


2241
NLpep154 (w/ Met)
A.A.
MSGVSGWRLFKKIS


 


2242
NLpep155 (w/ Met)
A.A.
MNSNVSGWRLFKKIS


 


2243
NLpep156 (w/ Met)
A.A.
MNSGVSGWRLFKKIS


 


2244
NLpep157 (w/ Met)
A.A.
MSVSGWRLFKKIS


 


2245
NLpep158 (w/ Met)
A.A.
MNSVSGWRLFKKIS


 


2246
NLpep159 (w/ Met)
A.A.
MSNVSGWRLFKKISN


 


2247
NLpep160 (w/ Met)
A.A.
MNSNVSGWRLFKKISN


 


2248
NLpep161 (w/ Met)
A.A.
MGWRLFKK


 


2249
NLpep162 (w/ Met)
A.A.
MGWALFKK


 


2250
NLpep163 (w/ Met)
A.A.
MVTGWALFEEIL


 


2251
NLpep164 (w/ Met)
A.A.
MVTGYALFQEIL


 


2252
NLpep165 (w/ Met)
A.A.
MVTGYALFEQIL


 


2253
NLpep166 (w/ Met)
A.A.
MVTGYALFEEIL


 


2254
NLpep167 (w/ Met)
N.A.
ATGGTGTCCGGCTGGGCACTGTTCAAGAAAATTTCC


 


2255
NLpep167 (w/ Met)
A.A.
MVSGWALFKKIS


 


2256
NLpep168 (w/ Met)
A.A.
MVSGWKLFKKIS


 


2257
NLpep169 (w/ Met)
N.A.
ATGGTGTCCGGCTGGCAGCTGTTCAAGAAAATTTCC


 


2258
NLpep169 (w/ Met)
A.A.
MVSGWQLFKKIS


 


2259
NLpep170 (w/ Met)
A.A.
MVSGWELFKKIS


 


2260
NLpep171 (w/ Met)
N.A.
ATGGTGTCCGGCTGGCTGCTGTTCAAGAAAATTTCC


 


2261
NLpep171 (w/ Met)
A.A.
MVSGWLLFKKIS


 


2262
NLpep172 (w/ Met)
N.A.
ATGGTGTCCGGCTGGGTGCTGTTCAAGAAAATTTCC


 


2263
NLpep172 (w/ Met)
A.A.
MVSGWVLFKKIS


 


2264
NLpep111 (w/o Met)
N.A.
GTGACCGGCTACCGGCTGTTCGAGGAGATCAGC


 


2265
NLpep111 (w/o Met)
A.A.
VTGYRLFEEIS


 


2266
NLpep112 (w/o Met)
N.A.
GTGACCGGCTACCGGCTGTTCGAGGAGGCCAGC


 


2267
NLpep112 (w/o Met)
A.A.
VTGYRLFEEAS


 


2268
NLpep113 (w/o Met)
N.A.
GTGACCGGCTACCGGCTGTTCGAGGAGGAGAGC


 


2269
NLpep113 (w/o Met)
A.A.
VTGYRLFEEES


 


2270
NLpep114 (w/o Met)
N.A.
GTGACCGGCTACCGGCTGTTCGAGGAGATCCTG


 


2271
NLpep114 (w/o Met)
A.A.
VTGYRLFEEIL


 


2272
NLpep115 (w/o Met)
N.A.
GTGACCGGCTACCGGCTGTTCGAGGAGGCCCTG


 


2273
NLpep115 (w/o Met)
A.A.
VTGYRLFEEAL


 


2274
NLpep116 (w/o Met)
N.A.
GTGACCGGCTACCGGCTGTTCGAGGAGGAGCTG


 


2275
NLpep116 (w/o Met)
A.A.
VTGYRLFEEEL


 


2276
NLpep117 (w/o Met)
N.A.
GTGGAGGGCTACCGGCTGTTCGAGGAGATCAGC


 


2277
NLpep117 (w/o Met)
A.A.
VEGYRLFEEIS


 


2278
NLpep118 (w/o Met)
N.A.
GTGGAGGGCTACCGGCTGTTCGAGGAGGCCAGC


 


2279
NLpep118 (w/o Met)
A.A.
VEGYRLFEEAS


 


2280
NLpep119 (w/o Met)
N.A.
GTGGAGGGCTACCGGCTGTTCGAGGAGGAGAGC


 


2281
NLpep119 (w/o Met)
A.A.
VEGYRLFEEES


 


2282
NLpep120 (w/o Met)
N.A.
GTGGAGGGCTACCGGCTGTTCGAGGAGATCCTG


 


2283
NLpep120 (w/o Met)
A.A.
VEGYRLFEEIL


 


2284
NLpep121 (w/o Met)
N.A.
GTGGAGGGCTACCGGCTGTTCGAGGAGGCCCTG


 


2285
NLpep121 (w/o Met)
A.A.
VEGYRLFEEAL


 


2286
NLpep122 (w/o Met)
N.A.
GTGGAGGGCTACCGGCTGTTCGAGGAGGAGCTG


 


2287
NLpep122 (w/o Met)
A.A.
VEGYRLFEEEL


 


2288
NLpep123 (w/o Met)
N.A.
GTGACCGGCTACCGGCTGTTCAAGAAGATCCTG


 


2289
NLpep123 (w/o Met)
A.A.
VTGYRLFKKIL


 


2290
NLpep124 (w/o Met)
N.A.
GTGACCGGCTACCGGCTGATGAAGAAGATCCTG


 


2291
NLpep124 (w/o Met)
A.A.
VTGYRLMKKIL


 


2292
NLpep125 (w/o Met)
N.A.
GTGACCGGCTACCGGCTGCACAAGAAGATCCTG


 


2293
NLpep125 (w/o Met)
A.A.
VTGYRLHKKIL


 


2294
NLpep126 (w/o Met)
N.A.
GTGACCGGCTACCGGCTGCTGAAGAAGATCCTG


 


2295
NLpep126 (w/o Met)
A.A.
VTGYRLLKKIL


 


2296
NLpep127 (w/o Met)
N.A.
GTGACCGGCTACCGGCTGAGCAAGAAGATCCTG


 


2297
NLpep127 (w/o Met)
A.A.
VTGYRLSKKIL


 


2298
NLpep128 (w/o Met)
N.A.
GTGACCGGCTACCGGCTGTTCGAGAAGATCCTG


 


2299
NLpep128 (w/o Met)
A.A.
VTGYRLFEKIL


 


2300
NLpep129 (w/o Met)
N.A.
GTGACCGGCTACCGGCTGATGGAGAAGATCCTG


 


2301
NLpep129 (w/o Met)
A.A.
VTGYRLMEKIL


 


2302
NLpep130 (w/o Met)
N.A.
GTGACCGGCTACCGGCTGCACGAGAAGATCCTG


 


2303
NLpep130 (w/o Met)
A.A.
VTGYRLHEKIL


 


2304
NLpep131 (w/o Met)
N.A.
GTGACCGGCTACCGGCTGCTGGAGAAGATCCTG


 


2305
NLpep131 (w/o Met)
A.A.
VTGYRLLEKIL


 


2306
NLpep132 (w/o Met)
N.A.
GTGACCGGCTACCGGCTGAGCGAGAAGATCCTG


 


2307
NLpep132 (w/o Met)
A.A.
VTGYRLSEKIL


 


2308
NLpep133 (w/o Met)
N.A.
GTGACCGGCTACCGGCTGATGGAGGAGATCCTG


 


2309
NLpep133 (w/o Met)
A.A.
VTGYRLMEEIL


 


2310
NLpep134 (w/o Met)
N.A.
GTGACCGGCTACCGGCTGCACGAGGAGATCCTG


 


2311
NLpep134 (w/o Met)
A.A.
VTGYRLHEEIL


 


2312
NLpep135 (w/o Met)
N.A.
GTGACCGGCTACCGGCTGCTGGAGGAGATCCTG


 


2313
NLpep135 (w/o Met)
A.A.
VTGYRLLEEIL


 


2314
NLpep136 (w/o Met)
N.A.
GTGACCGGCTACCGGCTGAGCGAGGAGATCCTG


 


2315
NLpep136 (w/o Met)
A.A.
VTGYRLSEEIL


 


2316
NLpep137 (w/o Met)
N.A.
GTGAGCGGCTACCGGCTGTTCGAGGAGATCCTG


 


2317
NLpep137 (w/o Met)
A.A.
VSGYRLFEEIL


 


2318
NLpep138 (w/o Met)
N.A.
GTGACCGGCTGGCGGCTGTTCGAGGAGATCCTG


 


2319
NLpep138 (w/o Met)
A.A.
VTGWRLFEEIL


 


2320
NLpep139 (w/o Met)
N.A.
GTGAGCGGCTGGCGGCTGTTCGAGGAGATCCTG


 


2321
NLpep139 (w/o Met)
A.A.
VSGWRLFEEIL


 


2322
NLpep140 (w/o Met)
N.A.
AACGTGACCGGCTACCGGCTGTTCGAGGAGATCCTG


 


2323
NLpep140 (w/o Met)
A.A.
NVTGYRLFEEIL


 


2324
NLpep141 (w/o Met)
N.A.
GTGACCGGCTACCGGCTGTTCGAGGAGATCCTGAAC


 


2325
NLpep141 (w/o Met)
A.A.
VTGYRLFEEILN


 


2326
NLpep142 (w/o Met)
N.A.
AACGTGACCGGCTACCGGCTGTTCGAGGAGATCCTG





AAC


 


2327
NLpep142 (w/o Met)
A.A.
NVTGYRLFEEILN


 


2328
NLpep143 (w/o Met)
N.A.
GTGACCGGCTACCGGCTGTTCGAGGAGATC


 


2329
NLpep143 (w/o Met)
A.A.
VTGYRLFEEI


 


2330
NLpep144 (w/o Met)
N.A.
GTGACCGGCTACCGGCTGTTCCAGAAGATCAGC


 


2331
NLpep144 (w/o Met)
A.A.
VTGYRLFQKIS


 


2332
NLpep145 (w/o Met)
N.A.
GTGACCGGCTACCGGCTGTTCAAGAAGATCAGCAAC


 


2333
NLpep145 (w/o Met)
A.A.
VTGYRLFKKISN


 


2334
NLpep146 (w/o Met)
N.A.
GTGACCGGCTACCGGCTGTTCAAGAAGATCAGC


 


2335
NLpep146 (w/o Met)
A.A.
VTGYRLFKKIS


 


2336
NLpep147 (w/o Met)
A.A.
VSGWRLFKKISA


 


2337
NLpep148 (w/o Met)
A.A.
GVSGWRLFKKIS


 


2338
NLpep149 (w/o Met)
A.A.
SVSGWRLFKKISN


 


2339
NLpep150 (w/o Met)
A.A.
SVSGWRLFKKISA


 


2340
NLpep151 (w/o Met)
A.A.
NSVSGWRLFKKISA


 


2341
NLpep152 (w/o Met)
A.A.
NSVSGWRLFKKISN


 


2342
NLpep153 (w/o Met)
A.A.
SNVSGWRLFKKIS


 


2343
NLpep154 (w/o Met)
A.A.
SGVSGWRLFKKIS


 


2344
NLpep155 (w/o Met)
A.A.
NSNVSGWRLFKKIS


 


2345
NLpep156 (w/o Met)
A.A.
NSGVSGWRLFKKIS


 


2346
NLpep157 (w/o Met)
A.A.
SVSGWRLFKKIS


 


2347
NLpep158 (w/o Met)
A.A.
NSVSGWRLFKKIS


 


2348
NLpep159 (w/o Met)
A.A.
SNVSGWRLFKKISN


 


2349
NLpep160 (w/o Met)
A.A.
NSNVSGWRLFKKISN


 


2350
NLpep161 (w/o Met)
A.A.
GWRLFKK


 


2351
NLpep162 (w/o Met)
A.A.
GWALFKK


 


2352
NLpep163 (w/o Met)
A.A.
VTGWALFEEIL


 


2353
NLpep164 (w/o Met)
A.A.
VTGYALFQEIL


 


2354
NLpep165 (w/o Met)
A.A.
VTGYALFEQIL


 


2355
NLpep166 (w/o Met)
A.A.
VTGYALFEEIL


 


2356
NLpep167 (w/o Met)
N.A.
GTGTCCGGCTGGGCACTGTTCAAGAAAATTTCC


 


2357
NLpep167 (w/o Met)
A.A.
VSGWALFKKIS


 


2358
NLpep168 (w/o Met)
A.A.
VSGWKLFKKIS


 


2359
NLpep169 (w/o Met)
N.A.
GTGTCCGGCTGGCAGCTGTTCAAGAAAATTTCC


 


2360
NLpep169 (w/o Met)
A.A.
VSGWQLFKKIS


 


2361
NLpep170 (w/o Met)
A.A.
VSGWELFKKIS


 


2362
NLpep171 (w/o Met)
N.A.
GTGTCCGGCTGGCTGCTGTTCAAGAAAATTTCC


 


2363
NLpep171 (w/o Met)
A.A.
VSGWLLFKKIS


 


2364
NLpep172 (w/o Met)
N.A.
GTGTCCGGCTGGGTGCTGTTCAAGAAAATTTCC


 


2365
NLpep172 (w/o Met)
A.A.
VSGWVLFKKIS









In certain embodiments, a peptide from Table 1 is provided. In some embodiments, peptides comprise a single amino acid difference from GVTGWRLCKRISA (SEQ ID NO: 236) and/or any of the peptides listed in Table 1. In some embodiments, peptides comprise two or more (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, etc.) amino acid differences from GVTGWRLCKRISA (SEQ ID NO: 236) and/or any of the peptides listed in Table 1. In some embodiments, peptides are provided comprising one of the amino acid sequences of SEQ ID NOS: 3-438 and 2162-2365. In some embodiments, peptides are provided comprising one of the amino acid sequences of SEQ ID NOS: 3-438 and 2162-2365 with one or more additions, substitutions, and/or deletions. In some embodiments, a peptide or a portion thereof comprises greater than 70% sequence identity (e.g., 71%, 75%, 80%, 85%, 90%, 95%, 99%) with one or more of the amino acid sequence of SEQ ID NOS: 3-438 and 2162-2365. In some embodiments, nucleic acids are provided comprising one of the nucleic acid coding sequences of SEQ ID NOS: 3-438 and 2162-2365. In some embodiments, nucleic acids are provided comprising one of the nucleic acid sequences of SEQ ID NOS: 3-438 and 2162-2365 with one or more additions, substitutions, and/or deletions. In some embodiments, a nucleic acid or a portion thereof comprises greater than 70% sequence identity (e.g., 71%, 75%, 80%, 85%, 90%, 95%, 99%) with one or more of the nucleic acid sequence of SEQ ID NOS: 3-438 and 2162-2365. In some embodiments, nucleic acids are provided that code for one of the amino acid sequences of SEQ ID NOS: 3-438 and 2162-2365. In some embodiments, nucleic acids are provided that code for one of the amino acid sequences of SEQ ID NOS: 3-438 and 2162-2365 with one or more additions, substitutions, and/or deletions. In some embodiments, a nucleic acid is provided that codes for an amino acid with greater than 70% sequence identity (e.g., 71%, 75%, 80%, 85%, 90%, 95%, 99%) with one or more of the amino acid sequences of SEQ ID NOS: 3-438 and 2162-2365.
In certain embodiments, a nucleic acid from Table 1 is provided. In some embodiments, a nucleic acid encoding a peptide from Table 1 is provided. In some embodiments, a nucleic acid of the present invention codes for a peptide that comprises a single amino acid difference from MGVTGWRLCERILA (SEQ ID NO: 2) and/or any of the peptides listed in Table 1. In some embodiments, nucleic acids code for peptides comprising two or more (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, etc.) amino acid differences from MGVTGWRLCERILA (SEQ ID NO: 2) and/or any of the peptides listed in Table 1. In some embodiments, nucleic acids are provided comprising the sequence of one of the nucleic acids in Table 1. In some embodiments, nucleic acids are provided comprising one of the nucleic acids of Table 1 with one or more additions, substitutions, and/or deletions. In some embodiments, a nucleic acid or a portion thereof comprises greater than 70% sequence identity (e.g., 71%, 75%, 80%, 85%, 90%, 95%, 99%) with one or more of the nucleic acids of Table 1.
In some embodiments, non-luminescent polypeptides that find use in embodiments of the present invention include polypeptides with one or more amino acid substitutions, deletions, or additions from SEQ ID NO: 440. In some embodiments, the present invention provides polypeptides comprising one or more of amino acid sequences of Table 2, and/or nucleic acids comprising the nucleic acid sequences of Table 2 (which code for the polypeptide sequences of Table 2).









TABLE 2





SEQ




ID




NO
Polymer
ID

















441
N.A.
R11N


442
A.A
R11N


443
N.A.
T13I


444
A.A
T13I


445
N.A.
G15S


446
A.A
G15S


447
N.A.
L18Q


448
A.A
L18Q


449
N.A.
Q20K


450
A.A
Q20K


451
N.A.
V27M


452
A.A
V27M


453
N.A.
F31I


454
A.A
F31I


455
N.A.
F31L


456
A.A
F31L


457
N.A.
F31V


458
A.A
F31V


459
N.A.
Q32R


460
A.A
Q32R


461
N.A.
N33K


462
A.A
N33K


463
N.A.
N33R


464
A.A
N33R


465
N.A.
I56N


466
A.A
I56N


467
N.A.
V58A


468
A.A
V58A


469
N.A.
I59T


470
A.A
I59T


471
N.A.
G67S


472
A.A
G67S


473
N.A.
G67D


474
A.A
G67D


475
N.A.
K75E


476
A.A
K75E


477
N.A.
M106V


478
A.A
M106V


479
N.A.
M106I


480
A.A
M106I


481
N.A.
D108N


482
A.A
D108N


483
N.A.
R112Q


484
A.A
R112Q


485
N.A.
N144T


486
A.A
N144T


487
N.A.
L149M


488
A.A
L149M


489
N.A.
N156D


490
A.A
N156D


491
N.A.
N156S


492
A.A
N156S


493
N.A.
V157D


494
A.A
V157D


495
N.A.
V157S


496
A.A
V157S


497
N.A.
G8A


498
A.A
G8A


499
N.A.
G15A


500
A.A
G15A


501
N.A.
G25A


502
A.A
G25A


503
N.A.
G26A


504
A.A
G26A


505
N.A.
G35A


506
A.A
G35A


507
N.A.
G48A


508
A.A
G48A


509
N.A.
G51A


510
A.A
G51A


511
N.A.
G64A


512
A.A
G64A


513
N.A.
G67A


514
A.A
G67A


515
N.A.
G71A


516
A.A
G71A


517
N.A.
G95A


518
A.A
G95A


519
N.A.
G101A


520
A.A
G101A


521
N.A.
G111A


522
A.A
G111A


523
N.A.
G116A


524
A.A
G116A


525
N.A.
G122A


526
A.A
G122A


527
N.A.
G129A


528
A.A
G129A


529
N.A.
G134A


530
A.A
G134A


531
N.A.
G147A


532
A.A
G147A


533
N.A.
I54A


534
A.A
I54A


535
N.A.
5A1




(G15A/D19A/G35A/G51A/G67A)


536
A.A
5A1




(G15A/D19A/G35A/G51A/G67A)


537
N.A. 
4A1




(G15A/G35A/G67A/G71A)


538
A.A
4A1




(G15A/G35A/G67A/G71A)


539
N.A.
5A2




(G15A/G35A/G51A/G67A/G71A)


540
A.A
5A2




(G15A/G35A/G51A/G67A/G71A)


541
N.A.
5A2 + A15G


542
A.A
5A2 + A15G


543
N.A.
5A2 + A35G


544
A.A
5A2 + A35G


545
N.A.
5A2 + A51G


546
A.A
5A2 + A51G


547
N.A.
5A2 + A67G


548
A.A
5A2 + A67G


549
N.A.
5A2 + A71G


550
A.A
5A2 + A71G


551
N.A.
5A2 + R11A


552
A.A
5A2 + R11A


553
N.A.
5A2 + R11C


554
A.A
5A2 + R11C


555
N.A.
5A2 + R11D


556
A.A
5A2 + R11D


557
N.A.
5A2 + R11E


558
A.A
5A2 + R11E


559
N.A.
5A2 + R11F


560
A.A
5A2 + R11F


561
N.A.
5A2 + R11G


562
A.A
5A2 + R11G


563
N.A.
5A2 + R11H


564
A.A
5A2 + R11H


565
N.A.
5A2 + R11I


566
A.A
5A2 + R11I


567
N.A.
5A2 + R11K


568
A.A
5A2 + R11K


569
N.A.
5A2 + R11L


570
A.A
5A2 + R11L


571
N.A.
5A2 + R11M


572
A.A
5A2 + R11M


573
N.A.
5A2 + R11N


574
A.A
5A2 + R11N


575
N.A.
5A2 + R11P


576
A.A
5A2 + R11P


577
N.A.
5A2 + R11Q


578
A.A
5A2 + R11Q


579
N.A.
5A2 + R11S


580
A.A
5A2 + R11S


581
N.A.
5A2 + R11T


582
A.A
5A2 + R11T


583
N.A.
5A2 + R11V


584
A.A
5A2 + R11V


585
N.A.
5A2 + R11W


586
A.A
5A2 + R11W


587
N.A.
5A2 + R11Y


588
A.A
5A2 + R11Y


589
N.A.
5A2 + A15C


590
A.A
5A2 + A15C


591
N.A.
5A2 + A15D


592
A.A
5A2 + A15D


593
N.A.
5A2 + A15E


594
A.A
5A2 + A15E


595
N.A.
5A2 + A15F


596
A.A
5A2 + A15F


597
N.A.
5A2 + A15G


598
A.A
5A2 + A15G


599
N.A.
5A2 + A15H


600
A.A
5A2 + A15H


601
N.A.
5A2 + A15I


602
A.A
5A2 + A15I


603
N.A.
5A2 + A15K


604
A.A
5A2 + A15K


605
N.A.
5A2 + A15L


606
A.A
5A2 + A15L


607
N.A.
5A2 + A15M


608
A.A
5A2 + A15M


609
N.A.
5A2 + A15N


610
A.A
5A2 + A15N


611
N.A.
5A2 + A15P


612
A.A
5A2 + A15P


613
N.A.
5A2 + A15Q


614
A.A
5A2 + A15Q


615
N.A.
5A2 + A15R


616
A.A
5A2 + A15R


617
N.A.
5A2 + A15S


618
A.A
5A2 + A15S


619
N.A.
5A2 + A15T


620
A.A
5A2 + A15T


621
N.A.
5A2 + A15V


622
A.A
5A2 + A15V


623
N.A.
5A2 + A15W


624
A.A
5A2 + A15W


625
N.A.
5A2 + A15Y


626
A.A
5A2 + A15Y


627
N.A.
5A2 + L18A


628
A.A
5A2 + L18A


629
N.A.
5A2 + L18C


630
A.A
5A2 + L18C


631
N.A.
5A2 + L18D


632
A.A
5A2 + L18D


633
N.A.
5A2 + L18E


634
A.A
5A2 + L18E


635
N.A.
5A2 + L18F


636
A.A
5A2 + L18F


637
N.A.
5A2 + L18G


638
A.A
5A2 + L18G


639
N.A.
5A2 + L18H


640
A.A
5A2 + L18H


641
N.A.
5A2 + L18I


642
A.A
5A2 + L18I


643
N.A.
5A2 + L18K


644
A.A
5A2 + L18K


645
N.A.
5A2 + L18M


646
A.A
5A2 + L18M


647
N.A.
5A2 + L18N


648
A.A
5A2 + L18N


649
N.A.
5A2 + L18P


650
A.A
5A2 + L18P


651
N.A.
5A2 + L18Q


652
A.A
5A2 + L18Q


653
N.A.
5A2 + L18R


654
A.A
5A2 + L18R


655
N.A.
5A2 + L18S


656
A.A
5A2 + L18S


657
N.A.
5A2 + L18T


658
A.A
5A2 + L18T


659
N.A.
5A2 + L18V


660
A.A
5A2 + L18V


661
N.A.
5A2 + L18W


662
A.A
5A2 + L18W


663
N.A.
5A2 + L18Y


664
A.A
5A2 + L18Y


665
N.A.
5A2 + F31A


666
A.A
5A2 + F31A


667
N.A.
5A2 + F31C


668
A.A
5A2 + F31C


669
N.A.
5A2 + F31D


670
A.A
5A2 + F31D


671
N.A.
5A2 + F31E


672
A.A
5A2 + F31E


673
N.A.
5A2 + F31G


674
A.A
5A2 + F31G


675
N.A.
5A2 + F31H


676
A.A
5A2 + F31H


677
N.A.
5A2 + F31I


678
A.A
5A2 + F31I


679
N.A.
5A2 + F31K


680
A.A
5A2 + F31K


681
N.A.
5A2 + F31L


682
A.A
5A2 + F31L


683
N.A.
5A2 + F31M


684
A.A
5A2 + F31M


685
N.A.
5A2 + F31N


686
A.A
5A2 + F31N


687
N.A.
5A2 + F31P


688
A.A
5A2 + F31P


689
N.A.
5A2 + F31Q


690
A.A
5A2 + F31Q


691
N.A.
5A2 + F31R


692
A.A
5A2 + F31R


693
N.A.
5A2 + F31S


694
A.A
5A2 + F31S


695
N.A.
5A2 + F31T


696
A.A
5A2 + F31T


697
N.A.
5A2 + F31V


698
A.A
5A2 + F31V


699
N.A.
5A2 + F31W


700
A.A
5A2 + F31W


701
N.A.
5A2 + F31Y


702
A.A
5A2 + F31Y


703
N.A.
5A2 + V58A


704
A.A
5A2 + V58A


705
N.A.
5A2 + V58C


706
A.A
5A2 + V58C


707
N.A.
5A2 + V58D


708
A.A
5A2 + V58D


709
N.A.
5A2 + V58E


710
A.A
5A2 + V58E


711
N.A.
5A2 + V58F


712
A.A
5A2 + V58F


713
N.A.
5A2 + V58G


714
A.A
5A2 + V58G


715
N.A.
5A2 + V58H


716
A.A
5A2 + V58H


717
N.A.
5A2 + V58I


718
A.A
5A2 + V58I


719
N.A.
5A2 + V58K


720
A.A
5A2 + V58K


721
N.A.
5A2 + V58L


722
A.A
5A2 + V58L


723
N.A.
5A2 + V58M


724
A.A
5A2 + V58M


725
N.A.
5A2 + V58N


726
A.A
5A2 + V58N


727
N.A.
5A2 + V58P


728
A.A
5A2 + V58P


729
N.A.
5A2 + V58Q


730
A.A
5A2 + V58Q


731
N.A.
5A2 + V58R


732
A.A
5A2 + V58R


733
N.A.
5A2 + V58S


734
A.A
5A2 + V58S


735
N.A.
5A2 + V58T


736
A.A
5A2 + V58T


737
N.A.
5A2 + V58W


738
A.A
5A2 + V58W


739
N.A.
5A2 + V58Y


740
A.A
5A2 + V58Y


741
N.A.
5A2 + A67C


742
A.A
5A2 + A67C


743
N.A.
5A2 + A67D


744
A.A
5A2 + A67D


745
N.A.
5A2 + A67E


746
A.A
5A2 + A67E


747
N.A.
5A2 + A67F


748
A.A
5A2 + A67F


749
N.A.
5A2 + A67G


750
A.A
5A2 + A67G


751
N.A.
5A2 + A67H


752
A.A
5A2 + A67H


753
N.A.
5A2 + A67I


754
A.A
5A2 + A67I


755
N.A.
5A2 + A67K


756
A.A
5A2 + A67K


757
N.A.
5A2 + A67L


758
A.A
5A2 + A67L


759
N.A.
5A2 + A67M


760
A.A
5A2 + A67M


761
N.A.
5A2 + A67N


762
A.A
5A2 + A67N


763
N.A.
5A2 + A67P


764
A.A
5A2 + A67P


765
N.A.
5A2 + A67Q


766
A.A
5A2 + A67Q


767
N.A.
5A2 + A67R


768
A.A
5A2 + A67R


769
N.A.
5A2 + A67S


770
A.A
5A2 + A67S


771
N.A.
5A2 + A67T


772
A.A
5A2 + A67T


773
N.A.
5A2 + A67V


774
A.A
5A2 + A67V


775
N.A.
5A2 + A67W


776
A.A
5A2 + A67W


777
N.A.
5A2 + A67Y


778
A.A
5A2 + A67Y


779
N.A.
5A2 + M106A


780
A.A
5A2 + M106A


781
N.A.
5A2 + M106C


782
A.A
5A2 + M106C


783
N.A.
5A2 + M106D


784
A.A
5A2 + M106D


785
N.A.
5A2 + M106E


786
A.A
5A2 + M106E


787
N.A.
5A2 + M106F


788
A.A
5A2 + M106F


789
N.A.
5A2 + M106G


790
A.A
5A2 + M106G


791
N.A.
5A2 + M106H


792
A.A
5A2 + M106H


793
N.A.
5A2 + M106I


794
A.A
5A2 + M106I


795
N.A.
5A2 + M106K


796
A.A
5A2 + M106K


797
N.A.
5A2 + M106L


798
A.A
5A2 + M106L


799
N.A.
5A2 + M106N


800
A.A
5A2 + M106N


801
N.A.
5A2 + M106P


802
A.A
5A2 + M106P


803
N.A.
5A2 + M106Q


804
A.A
5A2 + M106Q


805
N.A.
5A2 + M106R


806
A.A
5A2 + M106R


807
N.A.
5A2 + M106S


808
A.A
5A2 + M106S


809
N.A.
5A2 + M106T


810
A.A
5A2 + M106T


811
N.A.
5A2 + M106V


812
A.A
5A2 + M106V


813
N.A.
5A2 + M106W


814
A.A
5A2 + M106W


815
N.A. 
5A2 + M106Y


816
A.A
5A2 + M106Y


817
N.A. 
5A2 + L149A


818
A.A
5A2 + L149A


819
N.A. 
5A2 + L149C


820
A.A
5A2 + L149C


821
N.A.
5A2 + L149D


822
A.A
5A2 + L149D


823
N.A.
5A2 + L149E


824
A.A
5A2 + L149E


825
N.A.
5A2 + L149F


826
A.A
5A2 + L149F


827
N.A.
5A2 + L149G


828
A.A
5A2 + L149G


829
N.A.
5A2 + L149H


830
A.A
5A2 + L149H


831
N.A.
5A2 + L149I


832
A.A
5A2 + L149I


833
N.A.
5A2 + L149K


834
A.A
5A2 + L149K


835
N.A.
5A2 + L149M


836
A.A
5A2 + L149M


837
N.A.
5A2 + L149N


838
A.A
5A2 + L149N


839
N.A.
5A2 + L149P


840
A.A
5A2 + L149P


841
N.A.
5A2 + L149Q


842
A.A
5A2 + L149Q


843
N.A.
5A2 + L149R


844
A.A
5A2 + L149R


845
N.A.
5A2 + L149S


846
A.A
5A2 + L149S


847
N.A.
5A2 + L149T


848
A.A
5A2 + L149T


849
N.A.
5A2 + L149V


850
A.A
5A2 + L149V


851
N.A.
5A2 + L149W


852
A.A
5A2 + L149W


853
N.A.
5A2 + L149Y


854
A.A
5A2 + L149Y


855
N.A.
5A2 + V157A


856
A.A
5A2 + V157A


857
N.A.
5A2 + V157C


858
A.A
5A2 + V157C


859
N.A.
5A2 + V157D


860
A.A
5A2 + V157D


861
N.A.
5A2 + V157E


862
A.A
5A2 + V157E


863
N.A.
5A2 + V157F


864
A.A
5A2 + V157F


865
N.A.
5A2 + V157G


866
A.A
5A2 + V157G


867
N.A.
5A2 + V157H


868
A.A
5A2 + V157H


869
N.A.
5A2 + V157I


870
A.A
5A2 + V157I


871
N.A.
5A2 + V157K


872
A.A
5A2 + V157K


873
N.A.
5A2 + V157L


874
A.A
5A2 + V157L


875
N.A.
5A2 + V157M


876
A.A
5A2 + V157M


877
N.A.
5A2 + V157N


878
A.A
5A2 + V157N


879
N.A.
5A2 + V157P


880
A.A
5A2 + V157P


881
N.A.
5A2 + V157Q


882
A.A
5A2 + V157Q


883
N.A.
5A2 + V157R


884
A.A
5A2 + V157R


885
N.A.
5A2 + V157S


886
A.A
5A2 + V157S


887
N.A.
5A2 + V157T


888
A.A
5A2 + V157T


889
N.A.
5A2 + V157W


890
A.A
5A2 + V157W


891
N.A.
5A2 + V157Y


892
A.A
5A2 + V157Y


893
N.A.
5A2 + Q20K


894
A.A
5A2 + Q20K


895
N.A.
5A2 + V27M


896
A.A
5A2 + V27M


897
N.A.
5A2 + N33K


898
A.A
5A2 + N33K


899
N.A.
5A2 + V38I


900
A.A
5A2 + V38I


901
N.A.
5A2 + I56N


902
A.A
5A2 + I56N


903
N.A.
5A2 + D108N


904
A.A
5A2 + D108N


905
N.A.
5A2 + N144T


906
A.A
5A2 + N144T


907
N.A.
5A2 + V27M + A35G


908
A.A
5A2 + V27M + A35G


909
N.A.
5A2 + A71G + K75E


910
A.A
5A2 + A71G + K75E


911
N.A.
5A2 + R11E + L149M


912
A.A
5A2 + R11E + L149M


913
N.A.
5A2 + R11E + V157P


914
A.A
5A2 + R11E + V157P


915
N.A.
5A2 + D108N + N144T


916
A.A
5A2 + D108N + N144T


917
N.A.
5A2 + L149M + V157D


918
A.A
5A2 + L149M + V157D


919
N.A.
5A2 + L149M + V157P


920
A.A
5A2 + L149M + V157P


921
N.A.
3P




(5A2 + R11E + 




L149M + V157P)


922
A.A
3P




(5A2 + R11E + 




L149M + V157P)


923
N.A.
3P + D108N


924
A.A
3P + D108N


925
N.A.
3P + N144T


926
A.A
3P + N144T


927
N.A.
3E




(5A2 + R11E + 




L149M + V157E)


928
A.A
3E




(5A2 + R11E + 




L149M + V157E)


929
N.A.
3E + D108N


930
A.A
3E + D108N


931
N.A.
3E + N144T


932
A.A
3E + N144T


933
N.A.
5P




(3P + D108N + N144T)


934
A.A
5P




(3P + D108N + N144T)


935
N.A.
(5P + I65P6N)


936
A.A
(5P + I65P6N)


937
N.A.
5E




(3E + D108N + N144T)


938
A.A
5E




(3E + D108N + N144T)


939
N.A.
6E




(5E + I56N)


940
A.A
6E




(5E + I56N)


941
N.A.
NLpoly1




(5A2 + R11N + A15S + 




L18Q + F311 + V58A + 




A67D + M106V + 




L149M + V157D)


942
A.A
NLpoly1




(5A2 + R11N + A15S + 




L18Q + F311 + V58A + 




A67D + M106V + 




L149M + V157D)


943
N.A.
NLpoly2




(5A2 + A15S + L18Q + 




F31I + V58A + A67D + 




M106V + L149M + 




V157D)


944
A.A
NLpoly2




(5A2 + A15S + L18Q + 




F31I + V58A + A67D + 




M106V + L149M + 




V157D)


945
N.A.
NLpoly3




(5A2 + R11N + L18Q + 




F31I + V58A + A67D + 




M106V + L149M + 




V157D)


946
A.A
NLpoly3




(5A2 + R11N + L18Q + 




F31I + V58A + A67D + 




M106V + L149M + 




V157D)


947
N.A.
NLpoly4




(5A2 + R11N + A15S + 




F31I + V58A + A67D + 




M106V + L149M + 




V157D)


948
A.A
NLpoly4




(5A2 + R11N + A15S + 




F31I + V58A + A67D + 




M106V + L149M + 




V157D)


949
N.A.
NLpoly5




(5A2 + R11N + A15S + 




L18Q + V58A + A67D + 




M106V + L149M + 




V157D)


950
A.A
NLpoly5




(5A2 + R11N + A15S + 




L18Q + V58A + A67D + 




M106V + L149M + 




V157D)


951
N.A.
NLpoly6




(5A2 + R11N + A15S + 




L18Q + F31I + A67D + 




M106V + L149M + 




V157D)


952
A.A
NLpoly6




(5A2 + R11N + A15S + 




L18Q + F31I + A67D + 




M106V + L149M + 




V157D)


953
N.A.
NLpoly7




(5A2 + R11N + A15S + 




L18Q + F31I + V58A + 




M106V + L149M + 




V157D)


954
A.A
NLpoly7




(5A2 + R11N + A15S + 




L18Q + F31I + V58A + 




M106V + L149M + 




V157D)


955
N.A.
NLpoly8




(5A2 + R11N+ A15S + 




L18Q + F31I + V58A + 




A67D + L149M + 




V157D)


956
A.A
NLpoly8




(5A2 + R11N + A15S + 




L18Q + F31I + V58A + 




A67D + L149M + 




V157D)


957
N.A.
NLpoly9




(5A2 + R11N + A15S + 




L18Q + F31I + V58A + 




A67D + M106V + 




V157D)


958
A.A
NLpoly9




(5A2 + R11N + A15S + 




L18Q + F31I + V58A + 




A67D + M106V + 




V157D)


959
N.A.
NLpoly10




(5A2 + R11N + A15S + 




L18Q + F31I + V58A + 




A67D + M106V + 




L149M)


960
A.A
NLpoly10




(5A2 + R11N + A15S + 




L18Q + F31I + V58A + 




A67D + M106V + 




L149M)


961
N.A.
NLpoly11




(5A2 + A15S + L18Q + 




M106V + L149M + 




V157D)


962
A.A
NLpoly11




(5A2 + A15S + L18Q + 




M106V + L149M + 




V157D)


963
N.A.
NLpoly12




(5A2 + A15S + L18Q + 




A67D + M106V + 




L149M + V157D)


964
A.A
NLpoly12




(5A2 + A15S + L18Q + 




A67D + M106V + 




L149M + V157D)


965
N.A.
NLpoly13




(5A2 + R11N + A15S + 




L18Q + M106V + 




L149M + V157D)


966
A.A
NLpoly13




(5A2 + R11N + A15S + 




L18Q + M106V + 




L149M + V157D)


967
N.A.
5P + V


968
A.A
5P + V


969
N.A.
5P + A


970
A.A
5P + A


971
N.A.
5P + VT


972
A.A
5P + VT


973
N.A.
5P + VA


974
A.A
5P + VA


975 
N.A.
5P + AT


976 
A.A
5P + AT


977 
N.A.
5P + AA


978 
A.A
5P + AA


979 
N.A.
5P + GG


980 
A.A
5P + GG


981 
N.A.
5P + AA


982 
A.A
5P + AA


983 
N.A.
5P + ATG


984 
A.A
5P + ATG


985 
N.A.
5P + VTG


986 
A.A
5P + VTG


987 
N.A.
5P + VTA


988 
A.A
5P + VTA


989 
N.A.
5P + GTA


990 
A.A
5P + GTA


991
N.A.
5P + VTGW


992
A.A
5P + VTGW


993
N.A.
5P + VTGWR


994
A.A
5P + VTGWR


995
N.A.
5P + VTGWE


996
A.A
5P + VTGWE


997
N.A.
5P + VTGWK


998
A.A
5P + VTGWK


999
N.A.
5P + VTGWQ


1000
A.A
5P + VTGWQ


1001
N.A.
5P + VTGWH


1002
A.A
5P + VTGWH


1003
N.A.
5P D1 (−157)


1004
A.A
5P D1 (−157)


1005
N.A.
5P D2 (−156 − 157)


1006
A.A
5P D2 (−156 − 157)


1007
N.A.
5P D3 (−155 − 157)


1008
A.A
5P D3 (−155 − 157)


1009
N.A.
5P D4 (−154 − 157)


1010
A.A
5P D4 (−154 − 157)


1011
N.A.
5P D5 (−153 − 157)


1012
A.A
5P D5 (−153 − 157)


1013
N.A. 
5P D6 (−152 − 157)


1014
A.A
5P D6 (−152 − 157)


1015
N.A. 
5P D7 (−151 − 157)


1016
A.A
5P D7 (−151 − 157)


1017
N.A.
5P + F31A


1018
A.A
5P + F31A


1019
N.A.
5P + F31C


1020
A.A
5P + F31C


1021
N.A.
5P + F31D


1022
A.A
5P + F31D


1023
N.A.
5P + F31E


1024
A.A
5P + F31E


1025
N.A.
5P + F31G


1026
A.A
5P + F31G


1027
N.A.
5P + F31H


1028
A.A
5P + F31H


1029
N.A.
5P + F31I


1030
A.A
5P + F31I


1031
N.A.
5P + F31K


1032
A.A
5P + F31K


1033
N.A.
5P + F31L





1034
A.A
5P + F31L


1035
N.A.
5P + F31M


1036
A.A
5P + F31M


1037
N.A.
5P + F31N


1038
A.A
5P + F31N


1039
N.A.
5P + F31P


1040
A.A
5P + F31P


1041
N.A.
5P + F31Q


1042
A.A
5P + F31Q


1043
N.A.
5P + F31R


1044
A.A
5P + F31R


1045
N.A.
5P + F31S


1046
A.A
5P + F31S


1047
N.A.
5P + F31T


1048
A.A
5P + F31T


1049
N.A.
5P + F31V


1050
A.A
5P + F31V


1051
N.A.
5P + F31W


1052
A.A
5P + F31W


1053
N.A.
5P + F31Y


1054
A.A
5P + F31Y


1055
N.A.
5P + L46A


1056
A.A
5P + L46A


1057
N.A.
5P + L46C


1058
A.A
5P + L46C


1059
N.A.
5P + L46D


1060
A.A
5P + L46D


1061
N.A.
5P + L46E


1062
A.A
5P + L46E


1063
N.A.
5P + L46F


1064
A.A
5P + L46F


1065
N.A.
5P + L46G


1066
A.A
5P + L46G


1067
N.A.
5P + L46H


1068
A.A
5P + L46H


1069
N.A.
5P + L46I


1070
A.A
5P + L46I


1071
N.A.
5P + L46K


1072
A.A
5P + L46K


1073
N.A.
5P + L46M


1074
A.A
5P + L46M


1075
N.A.
5P + L46N


1076
A.A
5P + L46N


1077
N.A.
5P + L46P


1078
A.A
5P + L46P


1079
N.A.
5P + L46Q


1080
A.A
5P + L46Q


1081
N.A.
5P + L46R


1082
A.A
5P + L46R


1083
N.A.
5P + L46S


1084
A.A
5P + L46S


1085
N.A.
5P + L46T


1086
A.A
5P + L46T


1087
N.A.
5P + L46V


1088
A.A
5P + L46V


1089
N.A.
5P + L46W


1090
A.A
5P + L46W


1091
N.A.
5P + L46Y


1092
A.A
5P + L46Y


1093
N.A.
5P + N108A


1094
A.A
5P + N108A


1095
N.A.
5P + N108C


1096
A.A
5P + N108C


1097
N.A.
5P + N108D


1098
A.A
5P + N108D


1099
N.A.
5P + N108E


1100
A.A
5P + N108E


1101
N.A.
5P + N108F


1102
A.A
5P + N108F


1103
N.A.
5P + N108G


1104
A.A
5P + N108G


1105
N.A.
5P + N108H


1106
A.A
5P + N108H


1107
N.A.
5P + N108I


1108
A.A
5P + N108I


1109
N.A.
5P + N108K


1110
A.A
5P + N108K


1111
N.A.
5P + N108L


1112
A.A
5P + N108L


1113
N.A.
5P + N108M


1114
A.A
5P + N108M


1115
N.A.
5P + N108P


1116
A.A
5P + N108P


1117
N.A.
5P + N108Q


1118
A.A
5P + N108Q


1119
N.A.
5P + N108R


1120
A.A
5P + N108R


1121
N.A.
5P + N108S


1122
A.A
5P + N108S


1123
N.A.
5P + N108T


1124
A.A
5P + N108T


1125
N.A.
5P + N108V


1126
A.A
5P + N108V


1127
N.A.
5P + N108W


1128
A.A
5P + N108W


1129
N.A.
5P + N108Y


1130
A.A
5P + N108Y


1131
N.A.
5P + T144A


1132
A.A
5P + T144A


1133
N.A.
5P + T144C


1134
A.A
5P + T144C


1135
N.A.
5P + T144D


1136
A.A
5P + T144D


1137
N.A.
5P + T144E


1138
A.A
5P + T144E


1139
N.A.
5P + T144F


1140
A.A
5P + T144F


1141
N.A.
5P + T144G


1142
A.A
5P + T144G


1143
N.A.
5P + T144H


1144
A.A
5P + T144H


1145
N.A.
5P + T144I


1146
A.A
5P + T144I


1147
N.A.
5P + T144K


1148
A.A
5P + T144K


1149
N.A.
5P + T144L


1150
A.A
5P + T144L


1151
N.A.
5P + T144M


1152
A.A
5P + T144M


1153
N.A.
5P + T144N


1154
A.A
5P + T144N


1155
N.A.
5P + T144P


1156
A.A
5P + T144P


1157
N.A.
5P + T144Q


1158
A.A
5P + T144Q


1159
N.A.
5P + T144R


1160
A.A
5P + T144R


1161
N.A.
5P + T144S


1440
A.A
5P + T144S


1163
N.A.
5P + T144V


1164
A.A
5P + T144V


1165
N.A.
5P + T144W


1166
A.A
5P + T144W


1167
N.A.
5P + T144Y


1168
A.A
5P + T144Y


1169
N.A.
5P + P157A


1170
A.A
5P + P157A


1171
N.A.
5P + P157C


1172
A.A
5P + P157C


1173
N.A.
5P + P157D


1174
A.A
5P + P157D


1175
N.A.
5P + P157E


1176
A.A
5P + P157E


1177
N.A.
5P + P157F


1178
A.A
5P + P157F


1179
N.A.
5P + P157G


1180
A.A
5P + P157G


1181
N.A.
5P + P157H


1182
A.A
5P + P157H


1183
N.A.
5P + P157I


1184
A.A
5P + P157I


1185
N.A.
5P + P157K


1186
A.A
5P + P157K


1187
N.A.
5P + P157L


1188
A.A
5P + P157L


1189
N.A.
5P + P157M


1190
A.A
5P + P157M


1191
N.A.
5P + P157N


1192
A.A
5P + P157N


1193
N.A.
5P + P157Q


1194
A.A
5P + P157Q


1195
N.A.
5P + P157R


1196
A.A
5P + P157R


1197
N.A.
5P + P157S


1198
A.A
5P + P157S


1199
N.A.
5P + P157T


1200
A.A
5P + P157T


1201
N.A.
5P + P157V


1202
A.A
5P + P157V


1203
N.A.
5P + P157W


1204
A.A
5P + P157W


1205
N.A.
5P + P157Y


1206
A.A
5P + P157Y


1207
N.A.
5P + I107L


1208
A.A
5P + I107L


1209
N.A.
5P + K75E


1210
A.A
5P + K75E


1211
N.A.
5P + K123E + N156D


1212
A.A
5P + K123E + N156D


1213
N.A.
5P + I76V


1214
A.A
5P + I76V


1215
N.A.
5P + G48D + H57R +




L92M + I99V


1216
A.A
5P + G48D + H57R +




L92M + I99V


1217
N.A.
5P+ F31L + V36A + 




I99V


1218
A.A
5P + F31L + V36A +




I99V


1219
N.A.
5P + F31L + H93P


1220
A.A
5P + F31L + H93P


1221
N.A.
5P + V90A


1222
A.A
5P + V90A


1223
N.A.
5P + I44V


1224
A.A
5P + I44V


1225
N.A.
5P + L46R + H86Q +




M106V


1226
A.A
5P + L46R + H86Q +




M106V


1227
N.A.
5P + R141H


1228
A.A
5P + R141H


1229
N.A.
5P + N33D + V58A


1230
A.A
5P + N33D + V58A


1231
N.A.
5P + I56N + P157H


1232
A.A
5P + I56N + P157H


1233
N.A.
5P + L46Q + P157H


1234
A.A
5P + L46Q + P157H


1235
N.A.
5P + I59V


1236
A.A
5P + I59V


1237
N.A.
5P + A51T + E74K +




P113L


1238
A.A
5P + A51T + E74K +




P113L


1239
N.A.
5P + V36A


1240
A.A
5P + V36A


1241
N.A.
5P + A51T


1242
A.A
5P + A51T


1243
N.A.
5P + H57R


1244
A.A
5P + H57R


1245
N.A.
5P + V58A


1246
A.A
5P + V58A


1247
N.A.
5P + E74K


1248
A.A
5P + E74K


1249
N.A.
5P + H86Q


1250
A.A
5P + H86Q


1251
N.A.
5P + H93P


1252
A.A
5P + H93P


1253
N.A.
5P + I99V


1254
A.A
5P + I99V


1255
N.A.
5P + K123E


1256
A.A
5P + K123E


1257
N.A.
5P + T128S


1258
A.A
5P + T128S


1259
N.A.
5P + L142Q + T154N


1260
A.A
5P + L142Q + T154N


1261
N.A.
5P + H57Q


1262
A.A
5P + H57Q


1263
N.A.
5P + L92M


1264
A.A
5P + L92M


1265
N.A.
5P + P113L


1266
A.A
5P + P113L


1267
N.A.
5P + G48D


1268
A.A
5P + G48D


1269
N.A.
5P-B9 (−147 − 157)


1270
A.A
5P-B9 (−147 − 157)


1271
N.A.
5P + L46R + P157S


1272
A.A
5P + L46R + P157S


1273
N.A.
5P + L46H + P157H


1274
A.A
5P + L46H + P157H


1275
N.A.
5P + L46R + H93P


1276
A.A
5P + L46R + H93P


1277
N.A.
5P + L46R + H93P +




F31L


1278
A.A
5P + L46R + H93P +




F31L


1279
N.A.
5P + L46R + H93P +




K75E


1280
A.A
5P + L46R + H93P +




K75E


1281
N.A.
5P + L46R + H93P +




I76V


1282
A.A
5P + L46R + H93P +




I76V


1283
N.A.
8S




(5P + L46R + 




H93P + P157S + F31L)


1284
A.A
8S




(5P + L46R + H93P +




P157S + F31L)


1285
N.A.
5P + L46R + H93P +




P157S + K75E


1286
A.A
5P + L46R + H93P +




P157S + K75E


1287
N.A.
5P + L46R + H93P +




P157S + I76V


1288
A.A
5P + L46R + H93P +




P157S + I76V


1289
N.A.
12S




(8S + A51T + K75E +




I176V + I107L)


1290
A.A
12S




(8S + A51T + K75E +




I76V + I107L)


1291
N.A.
11S (12-A51T)


1292
A.A
11S (12-A51T)


1293
N.A.
12S-K75E


1294
A.A
12S-K75E


1295
N.A.
12S-I76V


1296
A.A
12S-I76V


1297
N.A.
12S-I107L


1298
A.A
12S-I107L









The polypeptides and coding nucleic acid sequences of Table 2 (SEQ ID NOS: 441-1298) all contain N-terminal Met residues (amino acids) or ATG start codons (nucleic acids). In some embodiments, the polypeptides and coding nucleic acid sequences of Table 2 are provided without N-terminal Met residues or ATG start codons (SEQ ID NOS: 1299-2156).
In certain embodiments, a polypeptide of one of the amino acid polymers of SEQ ID NOS: 441-2156 is provided. In some embodiments, polypeptides comprise a single amino acid difference from SEQ ID NO: 440. In some embodiments, polypeptides comprise two or more (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30 . . . 35 . . . 40 . . . 45 . . . 50, or more) amino acid differences from SEQ ID NO: 440 and/or any of the amino acid polymers of SEQ ID NOS:441-2156. In some embodiments, polypeptides are provided comprising the sequence of one of the amino acid polymers of SEQ ID NOS: 441-2156 with one or more additions, substitutions, and/or deletions. In some embodiments, a polypeptide or a portion thereof comprises greater than 70% sequence identity (e.g., >71%, >75%, >80%, >85%, >90%, >91%, >92%, >93%, >94%, >95%, >96%, >97%, >98%, or >99%) with one or more of the amino acid polymers of SEQ ID NOS: 441-2156.
In certain embodiments, a nucleic acid from Table 2 is provided. In some embodiments, a nucleic acid encoding a polypeptide from Table 2 is provided. In some embodiments, a nucleic acid of the present invention codes for a polypeptide that comprises a single amino acid difference from SEQ ID NO: 440 and/or any of the amino acid polymers of SEQ ID NOS: 441-2156. In some embodiments, nucleic acids code for a polypeptide comprising two or more (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30 . . . 35 . . . 40 . . . 45 . . . 50, or more) amino acid differences from SEQ ID NO: 440 and/or any of the polypeptides listed in Table 2. In some embodiments, nucleic acids are provided comprising the sequence of one of the nucleic acid polymers of SEQ ID NOS: 441-2156. In some embodiments, nucleic acids are provided comprising the sequence of one of the nucleic acid polymers of SEQ ID NOS: 441-2156 with one or more additions, substitutions, and/or deletions. In some embodiments, a nucleic acid or a portion thereof comprises greater than 70% sequence identity (e.g., >71%, >75%, >80%, >85%, >90%, >91%, >92%, >93%, >94%, >95%, >96%, >97%, >98%, or >99%) with one or more of the nucleic acid polymers of SEQ ID NOS: 441-2156. In some embodiments, a nucleic acid or a portion thereof codes for an polypeptide comprising greater than 70% sequence identity (e.g., >71%, >75%, >80%, >85%, >90%, >91%, >92%, >93%, >94%, >95%, >96%, >97%, >98%, or >99%) with one or more of the amino acid polymers of SEQ ID NOS: 441-2156. In some embodiments, nucleic acids are provided that code for one of the polypeptides of SEQ ID NOS: 441-2156. In some embodiments, nucleic acids are provided that code for one of the polypeptides of SEQ ID NOS: 441-2156 with one or more additions, substitutions, and/or deletions.
In some embodiments, a non-luminescent peptide or polypeptide and/or an interaction element, comprises a synthetic peptide, peptide containing one or more non-natural amino acids, peptide mimetic, conjugated synthetic peptide (e.g., conjugated to a functional group (e.g., fluorophore, luminescent substrate, etc.)).
The present invention provides compositions and methods that are useful in a variety of fields including basic research, medical research, molecular diagnostics, etc. Although the reagents and assays described herein are not limited to any particular applications, and any useful application should be viewed as being within the scope of the present invention, the following are exemplary assays, kits, fields, experimental set-ups, etc. that make use of the presently claimed invention.
Typical applications that make use of embodiments of the present invention involve the monitoring/detection of protein dimerization (e.g., heterodimers, homodimers), protein-protein interactions, protein-RNA interactions, protein-DNA interactions, nucleic acid hybridization, protein-small molecule interactions, or any other combinations of molecular entities. A first entity of interest is attached to a first member of a non-luminescent pair and the second entity of interest is attached to the second member of a non-luminescent pair. If a detectable signal is produced under the particular assay conditions, then interaction of the first and second entities are inferred. Such assays are useful for monitoring molecular interactions under any suitable conditions (e.g., in vitro, in vivo, in situ, whole animal, etc.), and find use in, for example, drug discovery, elucidating molecular pathways, studying equilibrium or kinetic aspects of complex assembly, high throughput screening, proximity sensor, etc.
In some embodiments, a non-luminescent pair of known characteristics (e.g., spectral characteristics, mutual affinity of pair) is used to elucidate the affinity of, or understand the interaction of, an interaction pair of interest. In other embodiments, a well-characterized interaction pair is used to determine the characteristics (e.g., spectral characteristics, mutual affinity of pair) of a non-luminescent pair.
Embodiments described herein may find use in drug screening and/or drug development. For example, the interaction of a small molecule drug or an entire library of small molecules with a target protein of interest (e.g., therapeutic target) is monitored under one or more relevant conditions (e.g., physiological conditions, disease conditions, etc.). In other embodiments, the ability of a small molecule drug or an entire library of small molecules to enhance or inhibit the interactions between two entities (e.g., receptor and ligand, protein-protein, etc.) is assayed. In some embodiments, drug screening applications are carried out in a high through-put format to allow for the detection of the binding of tens of thousands of different molecules to a target, or to test the effect of those molecules on the binding of other entities.
In some embodiments, the present invention provides the detection of molecular interactions in living organisms (e.g., bacteria, yeast, eukaryotes, mammals, primates, human, etc.) and/or cells. In some embodiments, fusion proteins comprising signal and interaction (target) polypeptides are co-expressed in the cell or whole organism, and signal is detected and correlated to the formation of the interaction complex. In some embodiments, cells are transiently and/or stably transformed or transfected with vector(s) coding for non-luminescent element(s), interaction element(s), fusion proteins (e.g., comprising a signal and interaction element), etc. In some embodiments, transgenic organisms are generated that code for the necessary fusion proteins for carrying out the assays described herein. In other embodiments, vectors are injected into whole organisms. In some embodiments, a transgenic animal or cell (e.g., expressing a fusion protein) is used to monitor the biodistribution of a small molecule or a biologic tethered (e.g., conjugated or genetically fused) to NLpeptide sequence that would form a complex in the subcellular compartments and/or tissues where it concentrates.
In some embodiments, a peptide (e.g., non-luminescent peptide) portion of a luminescent complex is employed as a protein tag (e.g., within cells). In such embodiments, a polypeptide (e.g., non-luminescent polypeptide) portion of a luminescent complex (e.g., capable of forming a luminescent complex with the non-luminescent peptide) is applied to cells (e.g., as part of a reagent) to detect/quantify the presence of proteins tagged with the non-luminescent peptide. For example, a protein of interest is fused to a high affinity NLpep (e.g., NLpep86). The NLpep is then transfected into cells of interest, a reagent containing NanoGlo+NLpoly11S is then added to cells+media, and luminescence is detected. This assay scheme is demonstrated in FIG. 175. In some embodiments, the small size of the peptide is useful for protein tagging. In some embodiments, non-luminescent polypeptides used in such a system are stable enough to exist in a suitable buffer for extended periods of time (e.g., in the presence of the furimazine substrate). In certain embodiments, the non-luminescent polypeptide has minimal detectable luminescence in the absence of the complementing peptide (e.g., even in the presence of furimazine substrate). In some embodiments, optimized buffer conditions are utilized to meet criteria necessary for protein tagging. High affinity spontaneously polypeptides and peptides are useful in such systems, and have utility in, for example, immunoassays, detection of virus particles, the study of protein dynamics in living cells, etc. In some embodiments, such a system provides an extremely small protein tag (e.g., 11 amino acids) providing high sensitivity detection, stability (e.g., particularly under denaturing conditions), and/or a broad dynamic range.
The compositions and methods provided herein, as well as any techniques or technologies based thereon find use in a variety of applications and fields, a non-limiting list of example applications follows:

    
    
        Antibody-free Western Blot: For example, a protein of interest is fused to a non-luminescent peptide (e.g., by genetic engineering) and expressed by any suitable means. The proteins separated (e.g., by PAGE) and transferred to a membrane. The membrane is then washed with complimentary non-luminescent polypeptide (e.g. allowing a luminescent complex to form), and placed on imager (e.g., utilizing a CCD camera) with Furimazine (PBI-3939) atop the membrane, and the protein of interest is detected (e.g., via the luminescence of the luminescent complex).
        “LucCytochemistry”: For example, a protein of interest is expressed fused to a non-luminescent peptide or polypeptide and then detected with a complimentary non-luminescent polypeptide or peptide in a fashion analogous to immunocytochemistry.
        Protein localization assay: For example, a localization signal is added to a non-luminescent polypeptide or polypeptide (e.g., via genetic engineering) and expressed in cells (e.g., a nuclear localization signal added would result in expression of the non-luminescent polypeptide in the nucleus). A complimentary non-luminescent peptide or polypeptide is fused to a protein of interest (e.g., via genetic engineering) and expressed in cells with the non-luminescent polypeptide or peptide. Luminescence is produced if the protein of interest localizes in the same subcellular compartment (e.g., the nucleus) as the signal-localized non-luminescent polypeptide.
        Protein Stability Assay: For example, a protein of interest is fused to a non-luminescent peptide or polypeptide (e.g., via genetic engineering) and incubated under one or more conditions of interest. A complimentary non-luminescent polypeptide or peptide is added (e.g., at various time points), and luminescence is used to quantify the amount of protein of interest (e.g., a proxy for stability).
        Protein Detection/Quantification: For example, a protein of interest fused to a non-luminescent peptide or polypeptide (e.g., via genetic engineering) and expressed and/or manipulated by any method. The complimentary non-luminescent polypeptide or peptide is then added to detect and/or quantify the protein of interest.
        Protein Purification: For example, a protein of interest is fused to a non-luminescent peptide or polypeptide (e.g., via genetic engineering) and expressed by any method. The mixture of proteins is passed through an immobilized complimentary non-luminescent polypeptide or peptide (e.g., on beads, on a column, on a chip, etc.), washed with suitable buffer and eluted (e.g., with a buffer of high ionic strength or low pH). A mutant form of the non-luminescent peptide or polypeptide that does not activate the luminescence of the complimentary non-luminescent peptide or polypeptide may be used to elute the protein of interest.
        Pull-down: For example, an immobilized, complimentary, non-luminescent polypeptide is used to isolate a protein of interest (and interacting proteins) that is fused to a non-luminescent peptide (e.g., via genetic engineering).
        G-Coupled Protein Receptor (GPCR) Internalization Assay: For example, a non-luminescent peptide or polypeptide is fused to a GPCR of interest (e.g., via genetic engineering) and expressed on the surface of cells. A complimentary non-luminescent polypeptide or peptide is added to the media of the cells and used to detect the GPCR on cell surface. A ligand is added to stimulate the internalization of the GPCR, and a decrease in luminescence is observed.
        Membrane Integrity Assay for Cell Viability: For example, when the cell membrane of a cell expressing a non-luminescent polypeptide become compromised, a non-luminescent peptide enters the cell (e.g., a peptide that otherwise can't cross the cell membrane), thereby forming a luminescent complex, and generating luminescence.
        5-Hydroxymethyl Cytosine Detection: For example, a cysteine is added to a non-luminescent peptide and incubated with DNA and a methyltransferase. The methyltransferase catalyzes the addition of the thiol (cysteine) only onto cytosine residues that are 5-hydroxymethylated. Unincorporated peptide is then separated from the DNA (using any method possible), and a non-luminescent polypeptide is added to detect the peptide conjugated to the DNA.
        Formyl Cytosine Detection: For example, similar to the 5-hydroxymethyl cytosine detection above, this detection method uses chemistry with specific reactivity for formyl cytosine.
        Viral Incorporation: Nucleic acid coding for a non-luminescent peptide or polypeptide is incorporated into a viral genome, and the complementary non-luminescent polypeptide or peptide is constitutively expressed in the target cells. Upon infection of the target cells and expression of the non-luminescent peptide, the bioluminescent complex forms and a signal is detected (e.g., in the presence of substrate).
        Chemical Labeling of Proteins: A non-luminescent peptide is fused or tethered to a reactive group (e.g., biotin, succinimidyl ester, maleimide, etc.). A protein of interest (e.g., antibody) is tagged with the non-luminescent peptide through binding of the reactive group to the protein of interest. Because the peptide is small, it does not affect the functionality of the protein of interest. Complimentary non-luminescent polypeptide is added to the system, and a luminescent complex is produced upon binding to the polypeptide to the peptide.
        Protease Assay: For example, a peptide sequence that is recognized by a protease of interest can be joined to NLPep in such a way that prevents bioluminescence upon exposure to NLPoly. Ways to do this include attaching a luminescence quencher to the protease recognition sequence or binding the protease recognition sequence to NLPep in such a way that complementation is hindered. Upon activity of the protease to cleave the recognition sequence, the ability of NLPoly to complement to NLPep and emit luminescence is restored, and thus the system is a sensitive protease assay.
        RNA detection.
        Biomolecule Linker characterization: For example, a linker attached to a biomolecule such as an antibody can be evaluated for its stability under a set of conditions through attaching NLPep to the molecule via the linker of interest. Over time, the production of free NLPep through linker degradation can be monitored by addition of NLPoly and furimazine and quantification of bioluminescence produced.
        Mutation assay: For example, a point mutation, a frameshift mutation, etc. introduced in vitro or in vivo results in either a gain of signal or loss of signal from a complementation pair. Such an assay could be used, for example, to test compounds for mutagenicity.
        Target engagement for peptide inhibitors: Use of low affinity NLpep-conjugated peptides (expressed in cells) to monitor target engagement of peptide-based inhibitors. NLpoly is tethered to the target of interest. Engagement results in loss of signal from luminescent complex.
        Gain of signal Protease biosensors: A protease cleavage site is expressed between NLpoly and a dark peptide NLpep (low affinity). Cleavage releases dark peptide allowing for high affinity NLpep to complement NLpoly.
        Gain of function protease assay: The sequence of an NLpep is engineered proximal to a cleavage site of a full length substrate for a protease (e.g., caspase, ADAM, etc). The peptide remains sterically inaccessible as long as the substrate remains intact and the peptide is “buried”. Both the genetically engineered protease substrate and a NLpoly (e.g., NLpoly11S) are co-transfected into a target cell line. Luciferase activity is induced upon induction of protease activity which leads to the cleavage of the substrate and exposure of the activator peptide on the N- or C-terminus of one of the fragments. This principle is expandable to detect conformational changes and/or protein modifications as well.
        Intracellular analyte quantification using recombinant intrabodies: Antibody fragments expressed within cells as NLpoly or NLpep fusion. Complementary subunit is genetically fused to an analyte of interest. When analyte is present, antibody binds and luminescent complex is formed. The application is expandable to intracellular PTM (e.g. phosphorylation) biosensors, in which the intrabody only binds to the analyte when it has been phosphorylated (or otherwise bound by the modification-specific Ab).

The above applications of the compositions and methods of the present invention are not limiting and may be modified in any suitable manner while still being within the scope of the present invention.

    
    


The present invention also provides methods for the design and/or optimization of non-luminescent pairs/groups and the bioluminescent complexes that form therefrom. Any suitable method for the design of non-luminescent pairs/groups that are consistent with embodiments described herein, and/or panels thereof, is within the scope of the present invention.
In certain embodiments, non-luminescent pairs/groups are designed de novo to lack luminescence individually and exhibit luminescence upon association. In such embodiments, the strength of the interaction between the non-luminescent elements is insufficient to produce a bioluminescent signal in the absence of interaction elements to facilitate formation of the bioluminescent complex.
In other embodiments, non-luminescent elements and/or non-luminescent pairs are rationally designed, for example, using a bioluminescent protein (e.g., SEQ ID NO: 2157) as a starting point. For example, such methods may comprise: (a) aligning the sequences of three or more related proteins; (b) determining a consensus sequence for the related proteins; (c) providing first and second fragments of a bioluminescent protein that is related to the ones from which the consensus sequence was determined, wherein the fragments are individually substantially non-luminescent but exhibit luminescence upon interaction of the fragments; (d) mutating the first and second fragments at one or more positions each (e.g., in vitro, in silico, etc.), wherein said mutations alter the sequences of the fragments to be more similar to a corresponding portion of the consensus sequence, wherein the mutating results in a non-luminescent pair that are not fragments of a preexisting protein, and (e) testing the non-luminescent pair for the absence of luminescence when unassociated and luminescence upon association of the non-luminescent pair. In other embodiments, first and second fragments of one of the proteins used in determining the consensus sequence are provided, mutated, and tested.
In some embodiments, a peptide of a luminescent pair is a ‘dark peptide,’ or one that binds to its complement (e.g., NLpoly) (e.g., with low or high affinity) but produces minimal or no luminescence (See FIGS. 180-182). In some embodiments, a high affinity dark peptide finds use in inverse complementation, or gain of signal assays for measuring inhibitors. In some embodiments, a low affinity dark peptide is used to bring down background of NLpoly11S in a reagent for the detection of a high affinity peptide tag (e.g. NLpep86). Exemplary dark peptides are provided in FIG. 180.
In some embodiments, a peptide of a luminescent pair is a ‘quencher peptide,’ or one that contains a quencher moiety (e.g., DAB), and the quencher absorbs the light/energy produced by both a NLpoly in isolation (e.g., the signal produced independent of a complementing NLpep) and a NLpoly-NLpep complex (e.g., the signal produced as a result of complex formation). Exemplary dark quencher peptides would have a suitable absorption spectrum and include DAB-161 (DAB-GWRLFKK(SEQ ID NO: 2370)), DAB-162 (DAB-GWALFKK (SEQ ID NO: 2351)), DAB-163 (DAB-VTGWALFEEIL(SEQ ID NO: 2372)), DAB-164 (DAB-VTGYALFQEIL (SEQ ID NO: 2573)), DAB-165 (DAB-VTGYALFEQIL (SEQ ID NO: 2574), and DAB-166 (DAB-VTGYALFEEIL (SEQ ID NO: 2575); wherein DAB=Dabcyl (475 nm quencher)+dPEG4 spacer.
In some embodiments, the above methods are not limited to the design and/or optimization of non-luminescent pairs. The same steps are performed to produce pairs of elements that lack a given functionality (e.g., enzymatic activity) individually, but display such functionality when associated. In any of these cases, the strength of the interaction between the non-luminescent pair elements may be altered via mutations to ensure that it is insufficient to produce functionality in the absence of interaction elements that facilitate formation of the bioluminescent complex.
EXPERIMENTAL
Example 1
Generation of Peptides
Peptide constructs were generated by one of three methods: annealing 5′-phosphorylated oligonucleotides followed by ligation to pF4Ag-Barnase-HALOTAG vector (Promega Corporation; cut with SgfI and XhoI) or pFN18A (Promega Corporation; cut with SgfI and XbaI), site directed mutagenesis using Quik Change Lightning Multi kit from Agilent or outsourcing the cloning to Gene Dynamics.
Example 2
Peptide Preparation
The peptides generated in Example 1 were prepared for analysis by inoculating a single colony of KRX E. coli cells (Promega Corporation) transformed with a plasmid encoding a peptide into 2-5 ml of LB culture and grown at 37° C. overnight. The overnight cultures (10 ml) were then diluted into 1 L of LB and grown at 37° C. for 3 hours. The cultures were then induced by adding 10 ml 20% rhamnose to the 1 L culture and induced at 25° C. for 18 hours.
After induction, 800 ml of each culture was spun at 5000×g at 4° C. for 30 minutes. The pellet generated was then resuspended in 80 ml Peptide Lysis Buffer (25 mM HEPES pH 7.4, 0.1× Passive Lysis Buffer (Promega Corporation), 1 ml/ml lysozyme and 0.03 U/μl RQ1 DNase (Promega Corporation)) and incubated at room temperature for 15 minutes. The lysed cells were then frozen on dry ice for 15 minutes and then thawed in a room temperature bath for 15 minutes. The cells were then spun at 3500×g at 4° C. for 30 minutes. The supernatants were aliquoted into 10 ml samples with one aliquot of 50 μl placed into a 1.5 ml tube.
To the 50 μl samples, 450 μl H2O and 167 μl 4×SDS Loading Dye were added, and the samples incubated at 95° C. for 5 minutes. After heating, 5 μl of each sample was loaded (in triplicate) onto an SDS-PAGE gel, and the gel run and stained according to the manufacturer's protocol. The gel was then scanned on a Typhoon Scanner (excitation 532 nm, emission 580 nm, PMT sensitivity 400V). The resulting bands were quantified using the ImageQuant (5.2) software. Each of the three replicate intensities was averaged, and the average intensity of NLpep53-HT was defined at 12× concentration. The concentrations of all other peptides were relative to Pep53-HT.
Example 3
Peptide Analysis
All of the peptides generated in Examples 1-2 contained single mutations to the peptide sequence: GVTGWRLCKRISA (SEQ ID NO: 236). All of the peptides were fused to a HALOTAG protein (Promega Corporation). Peptides identified as “HT-NLpep” indicate that the peptide is located at the C-terminus of the HALOTAG protein. In this case, the gene encoding the peptide includes a stop codon, but does not include a methionine to initiate translation. Peptides identified as “NLpep-HT” indicate that the peptide is at the N-terminus of the HALOTAG protein. In this case, the peptide does include a methionine to initiate translation, but does not include a stop codon.
To determine the ability of the peptides to activate luminescence, individual colonies of KRX E. coli cells (Promega Corporation) was transformed with a plasmid encoding a peptide from Example 1, inoculated in 200 μl of minimal medium (1×M9 salts, 0.1 mM CaCl2, 2 mM MgSO4, 1 mM Thiamine HCl, 1% gelatin, 0.2% glycerol, and 100 ul/ml Ampicillin) and grown at 37° C. overnight. In addition to the peptides, a culture of KRX E. coli cells expressing a wild-type (WT) fragment of residues 1-156 of the NanoLuc was grown. All peptides and the WT fragment were inoculated into at least 3 separate cultures.
After the first overnight growth, 10 μl of culture was diluted into 190 μl fresh minimal medium and again grown at 37° C. overnight.
After the second overnight growth, 10 μl of the culture was diluted into 190 μl of auto-induction medium (minimal medium+5% glucose and 2% rhamnose). The cultures were then inducted at 25° C. for approximately 18 hours.
After induction, the small peptide mutant cultures were assayed for activity. The cultures containing the WT 1-156 fragment were pooled, mixed with 10 ml of 2× Lysis Buffer (50 mM HEPES pH 7.4, 0.3× Passive Lysis Buffer, and 1 mg/ml lysozyme) and incubated at room temperature for 10 minutes. 30 μl of the lysed WT 1-156 culture was then aliquoted into wells of a white, round bottom 96-well assay plate (Costar 3355). To wells of the assay plate, 20 μl of a peptide culture was added, and the plate incubated at room temperature for 10 minutes. After incubation, 50 μl NANOGLO Luciferase Assay Reagent (Promega Corporation) was added, and the samples incubated at room temperature for 10 minutes. Luminescence was measured on a GLOMAX luminometer with 0.5 s integrations.
The results (See Table 3 and FIG. 1) demonstrate various mutations in the peptide (relative to SEQ ID NO: 1) that altered (e.g., increased, decreased) the luminescence following complementation with the wild-type non-luminescent polypeptide. The increased luminescence is thought to stem from one (or a combination) of five main factors, any of which are beneficial: affinity between the non-luminescent peptide and non-luminescent polypeptide, expression of the peptide, intracellular solubility, intracellular stability, and bioluminescent activity. The present invention though is not limited to any particular mechanism of action and an understanding of the mechanism of action is not necessary to practice the present invention.











TABLE 3





Mutation
HT-NLPep
NLpep-HT
HT-Pep st. dev.
Pep-HT st. dev.







G157D
0.1137
0.5493
N.D.
N.D.


G157N
0.6415
3.3074
0.2512
1.4828


G157S
1.9937
1.7156
0.8554
1.0563


G157E
0.1959
1.4461
0.0811
0.3221


G157H
0.9380
0.5733
0.4366
0.2277


G157C
N.D.
0.0468
N.D.
0.0081


G157P
N.D.
0.0543
N.D.
0.0106


V158I
0.6075
1.6010
0.3283
0.6264


V158A
0.1348
0.1438
0.0561
0.0447


V158K
0.0770
0.1923
0.0323
0.0521


V158Q
0.0445
0.0397
0.0188
0.0160


V158S
0.0487
0.0838
0.0189
0.0251


T159V
0.5658
0.0455
0.2293
0.0005


T159K
0.0490
0.0307
0.0120
0.0103


T159Q
0.3979
0.0310 
0.1063
0.0091


W161T
0.0028
0.0100
0.0007 
0.0049


W161K
0.0002
0.0008
9.7E-06
0.0001


W161V
0.0086
0.0050 
0.0062
0.0016


W161F
N.D.
0.0717 
N.D.
0.0049


W161Y
N.D.
0.2154
N.D.
0.0103


W161E
N.D.
0.0012
N.D.
0.0002


L163I
N.D.
0.2923
N.D.
0.1198


L163V
0.1727
0.1190
0.0257 
0.0288


L163T
0.0259
0.0262
0.0077 
0.0122


L163Y
0.0512
0.1959 
0.0126
0.1043


L163K
0.0885
0.0786 
0.0130
0.0244


C164N
0.0874
0.1081
0.0097 
0.0160


C164T
0.0116
0.0084
0.0029
0.0013


C164F
N.D.
13.3131 
N.D.
3.6429


C164Y
N.D.
1.0092 
N.D.
0.2592


C164S
N.D.
0.0202
N.D.
0.0029


C164H
N.D.
0.7597
N.D.
0.2149


C164M
N.D.
3.2618
N.D.
1.1763


C164A
N.D.
0.0858
N.D.
0.0196


C164Q
N.D.
0.0211
N.D.
0.0044


C164L
N.D.
1.0170
N.D.
0.2464


C164K
N.D.
0.0005
N.D.
0.0001


R166K
1.0910
1.2069
0.2266
0.5913


R166N
0.1033
0.1182
0.0289
0.0542


I167V
0.8770
1.0824
0.1113
0.2642


I167Q
0.0178
0.1172
0.0252
0.0150


I167E 
0.2771
0.2445
0.0358
0.0456


I167R
0.0464
0.0469
0.0027
0.0084


I167F
0.2832
0.1793
0.0159
0.0683


A169N 
0.9115
1.7775
0.1114
0.5901


A169T
0.9448
1.3720
0.0930
0.6021


A169R 
0.9851
0.5014
0.2205
0.1895


A169L 
1.1127
0.9047
0.1906
0.2481


A169E
0.8457
0.7889
0.1445
0.0819









Example 4
Generation of Non-Luminescent Polypeptides
Using pF4Ag-NanoLuc1-156 (WT 1-156) as a template, error-prone PCR (epPCR) was performed using the Diversify PCR Random Mutagenesis Kit from Clontech. The resulting PCR product was digested with SgfI and XbaI and ligated to pF4Ag-Barnase (Promega Corporation), a version of the commercially-available pF4A vector (Promega) which contains T7 and CMV promoters and was modified to contain an E. coli ribosome-binding site. Following transformation into KRX E. coli cells (Promega Corporation) by heat shock at 42° C., individual colonies were used to inoculate 200 μl cultures in clear, flat bottom 96-well plates (Costar 3370).
Example 5
Non-Luminescent Polypeptide Analysis
To determine the luminescence of the non-luminescent polypeptide mutants generated in Example 4, individual colonies of the KRX E. coli cells (Promega Corporation) transformed with a plasmid containing one of the non-luminescent polypeptide mutants from Example 4 was grown according to the procedure used in Example 3. The bacterial cultures were also induced according to the procedure used in Example 3.
To assay each non-luminescent polypeptide mutant induced culture, 30 μl of assay lysis buffer (25 mM HEPES pH 7.4, 0.3× Passive Lysis Buffer (Promega Corporation)), 0.006 U/μl RQ1 DNase (Promega Corporation) and 1× Peptide Solution (the relative concentration of the peptides were determined as explained in Example 2; from the relative concentration determined, the peptides were diluted to 1× in the lysis buffer) containing either the peptide fragment GVTGWRLCKRISA (SEQ ID NO: 18) or GVTGWRLFKRISA (SEQ ID NO: 106) were aliquoted into wells of a 96-well assay plate (Costar 3355). To the wells of the assay plate, 20 μl of an induced non-luminescent polypeptide mutant culture was added, and the plate incubated at room temperature for 10 minutes. After incubation, 50 μl of NANOGLO Luciferase Assay Reagent (Promega Corporation) was added, and the samples incubated at room temperature for 10 minutes. Luminescence was measured on a GLOMAX luminometer with 0.5 s integrations.
The results (Table 4 and FIG. 2) demonstrate numerous point mutations that improve the luminescence of the non-luminescent polypeptide upon complementation with two different peptides. Similar to the mutations in the peptide, these mutations in the non-luminescent polypeptide may stem from various factors, all of which are beneficial to the system as a whole.











TABLE 4







Mutation
V157D
F31I
L18Q
R11N





GVTGWRLCKRISA
4.98
4.1 
3.81
3.37


st dev
0.48
0.37
0.29
0.67


GVTGWRLFKRISA
3.02
2.83
2.99
2.09


st dev
0.77
0.61
0.82
0.03





Mutation
Q32R
M106V
M106I
G67S





GVTGWRLCKRISA
1.52
1.3 
1.27
1.22


st dev
0.2 
0.22
0.04
0.26


GVTGWRLFKRISA
1.04
1.4 
1.31
1.29


st dev
0.19
0.25
0.35
0.22





Mutation
F31L
L149M
N33K
I59T





GVTGWRLCKRISA
3.13
2.89
2.15
1.07


st dev
0.26
0.39
0.2 
0.07


GVTGWRLFKRISA
2.86
2.16
1.76
1.35


st dev
0.7 
0.26
0.08
0.37





Mutation
I56N
T13I
F31V
N33R





GVTGWRLCKRISA
0.44
2.18
2.12
2.1 


st dev
0.05
0.75
0.09
0.18


GVTGWRLFKRISA
1.81
1.44
2.12
1.56


st dev
0.35
0.34
0.46
0.16





Mutation
V27M
Q20K
V58A
K75E





GVTGWRLCKRISA
1.99
4.43
1.88
2.08


st dev
0.09
0.84
0.6 
0.47


GVTGWRLFKRISA
1.7 
2.33
1.07
2.05


st de
0.11
0.38
0.26
0.37





Mutation
G15S
G67D
R112N
N156D





GVTGWRLCKRIS
1.98
1.78
1.61
1.57


st de
0.99
0.11
0.2 
0.21


GVTGWRLFKRIS
2.34
1.57
1.45
1.21


st de
0.82
0.17
0.47
0.26





Mutation
D108N
N144T
N156S





GVTGWRLCKRIS
2.08
3.69
1.04



st de
0.6 
1.12
0.29



GVTGWRLFKRIS
1.88
2.26
1.4 



st de
0.38
0.51
0.28





*Units in Table 4 are RLU(mutant)/RLU(WT)






Example 6
Glycine to Alanine Substitutions in Non-Luminescent Polypeptide
The following example identified glycine residues within the non-luminescent polypeptide that can be substituted to alanine to provide an improved (e.g., greater luminescent signal) non-luminescent polypeptide. The substitutions were made singly (See FIG. 3), or in composites (FIG. 2). Non-luminescent polypeptides containing glycine to alanine substitutions were generated as described in Example 1.
Each single mutant colony was inoculated in 200 μl Minimal Media (1×M9 salts, 0.1 mM CaCl2, 2 mM MgSO4, 1 mM Thiamine HCl, 1% gelatin, 0.2% glycerol and 1× ampicillin) and incubated with shaking at 37° C. for 20 hours. 10 μl of the culture was then added to 190 μl of fresh Minimal Media and incubated again with shaking at 37° C. for 20 hours. 10 μl of the second culture was then added to 190 μl Auto-Induction Media (Minimal Media+5% glucose+2% rhamnose) and incubated with shaking at 25° C. for 18 hours to allow expression of the non-luminescent polypeptide.
To assay each mutant culture, 30 μl of assay lysis buffer (50 mM HEPES pH 7.5, 0.3× Passive Lysis Buffer (Promega Corporation)) and 0.006 U/μl RQ1 DNase (Promega Corporation)) containing non-luminescent peptide (1:10 dilution of NLpep9-HT (NLpep9 is SEQ ID NO: 17 and 18; HT is HaloTag E. coli clarified lysate) was added. The samples were shaken at room temperature for 10 minutes, and then 50 μl NANOGLO Luciferase Assay Reagent (Promega Corporation) was added. The samples were incubated at room temperature for 10 minutes, and luminescence was measured on a GLOMAX luminometer with 0.5 s integrations.
To generate the NLpep9-HT E. coli clarified lysate, 5 ml LB was inoculated with a single E. coli colony of NLpep9-HT and incubated at 37° C. overnight. 500 μl of the overnight culture was then diluted in 50 mls LB and incubated at 37° C. for 3 hours. 500 μl of 20% rhamnose was added and incubated at 25° C. for 18 hours. The expression culture was centrifuged at 3000×g for 30 minutes, and the cell pellet resuspended in 5 ml peptide lysis buffer (25 mM HEPES, pH 7.5, 0.1× Passive Lysis Buffer, 1 mg/ml lysozyme, and 0.3 U/μl RQ1 DNase) and incubated at room temperature for 10 minutes. The lysed sample was placed on dry ice for 15 minutes, thawed in a room temperature water bath and centrifuged at 3500×g for 30 minutes. The supernatant was the clarified lysate.
FIGS. 3 and 4 demonstrate the effects of the mutations on luminescence.
Example 7
Mutations in Non-Luminescent Peptide
In the following example, mutations were made in the non-luminescent peptide based on alignment to other fatty acid binding proteins (FABPs) and were chosen based on high probability (frequency in FABPs) to identify a mutation that retains/improves activity (such as NLpep2, 4, and 5) or establish that a mutation is not likely to be tolerated at that position (such as NLpep3). NLpep1-5 contain single mutations (See Table 1), and NLpep6-9 are composite sets of the mutations in NLpep2, 4, and 5 (See Table 1). Mutants were generated as described in Example 1.
Each mutant colony was inoculated in 200 μl Minimal Media and incubated with shaking at 37° C. for 20 hours. 10 μl of the culture was then added to 190 μl of fresh Minimal Media and incubated again with shaking at 37° C. for 20 hours. 10 μl of the second culture was then added to 190 μl Auto-Induction Media and incubated with shaking at 25° C. for 18 hours to allow expression of the non-luminescent peptide mutant.
To assay each mutant culture, 30 μl of assay lysis buffer (50 mM HEPES pH 7.5, 0.3× Passive Lysis Buffer (Promega Corporation)) and 0.006 U/μl RQ1 DNase (Promega Corporation)) containing non-luminescent polypeptide (1:10 dilution of wild-type non-luminescent polypeptide E. coli clarified lysate) was added. The samples were shaken at room temperature for 10 minutes, and then 50 μl NANOGLO Luciferase Assay Reagent (Promega Corporation) added. The samples were incubated at room temperature for 10 minutes, and luminescence was measured on a GLOMAX luminometer with 0.5 s integrations.
FIG. 1 shows the luminescence (RLUs) detected in each non-luminescent peptide mutant. The results demonstrate various positions that are able to tolerate a mutation without substantial loss in luminescence, as well as a few specific mutations that improve luminescence.
Example 8
Effect of Orientation of Fusion Tag on Luminescence
In the following example, luminescence generated by non-luminescent peptides with N- or C-terminus HaloTag protein was compared.
Single colony of each peptide-HT fusion was grown according to the procedure used in Example 7. The bacterial cultures were also induced according to the procedure used in Example 7. Luminescence was assayed and detected according to the procedure used in Example 7. FIGS. 6 and 7 demonstrate the luminescence (RLUs) detected in each peptide-HT fusion. The results demonstrate combinations of mutations that produce similar luminescence as NLpep1.
Example 9
Effect of Multiple Freeze-Thaw Cycles on Non-Luminescent Peptides
1 ml of NLpep9-HT was frozen on dry ice for 5 minutes and then thawed in a room temperature water bath for 5 minutes. 60 μl was then removed for assaying. The freeze-thaw procedure was then repeated another 10 times. After each freeze-thaw cycle, 60 μl of sample was removed for assaying.
To assay, 20 μl of each freeze-thaw sample was mixed with 30 μl of SEQ ID NO:2 and incubated at room temperature for 10 minutes. 50 μl of NANOGLO Luciferase Assay Reagent was added, and the samples incubated at room temperature for 10 minutes. Luminescence was measured on a GLOMAX luminometer with 0.5 s integrations. The results are depicted in FIG. 8 and demonstrate that NLpep can be subjected to multiple freeze-thaw cycles without a loss in activity (luminescence).
Example 10
Distinction of Mutations in Non-Luminescent Peptides
In the following example, TMR gel analysis was used to normalize the concentration of the non-luminescent peptide mutants to distinguish mutations that alter the expression from those that alter luminescence (e.g., altered luminescence may stem from altered binding affinity).
5 ml of LB was inoculated with a single mutant peptide colony and incubated with shaking at 37° C. for 20 hours. 50 μl of the overnight culture was diluted into 5 ml of fresh LB and incubated with shaking at 37° C. for 3 hours. 50 μl of 20% rhamnose was then added and incubated with shaking at 25° C. for 18 hours.
For TMR gel analysis, 79 μl of each induced culture was mixed with 10 μl 10× Fast Break Lysis Buffer (Promega Corporation), 10 μl of a 1:100 dilution of HALOTAG TMR ligand (Promega Corporation) non-luminescent polypeptide and 10 μl of RQ1 DNase and incubated at room temperature for 10 minutes. 33.3 μl of 4×SDS-loading buffer was added, and the samples incubated at 95° C. for 5 minutes. 15 μl of each sample was loaded onto an SDS gel and run according to the manufacturer's directions. The gel was then scanned on a Typhoon.
Each culture was diluted based on the TMR-gel intensity to normalize concentrations. 20 μl of each diluted culture was then mixed with 30 μl assay lysis buffer containing non-luminescent polypeptide (1:10 dilution of SEQ ID NO: 2 E. coli clarified lysate) and incubated with shaking at room temperature for 10 minutes. 50 μl of NANOGLO Luciferase Assay Reagent was added, and the samples incubated at room temperature for 10 minutes. Luminescence was measured on a GLOMAX luminometer with 0.5 s integrations (SEE FIG. 9).
Example 11
Site Saturation in Non-Luminescent Polypeptide
In the following example, positions 11, 15, 18, 31, 58, 67, 106, 149, and 157 were identified as sites of interest from screening the library of random mutations in wild-type non-luminescent polypeptide. All 20 amino acids at these positions (built on 5A2 non-luminescent mutant generated in Example 6 (SEQ ID NOS: 539 and 540) to validate with other mutations in the 5A2 mutant) were compared to determine the optimal amino acid at that position. Mutant non-luminescent polypeptides were generated as previously described in Example 1. Single colony of each non-luminescent polypeptide mutant was grown according to the procedure used in Example 6. The bacterial cultures were also induced according to the procedure used in Example 6. Luminescence was assayed and detected according to the procedure used in Example 6 expect NLpep53 E. coli clarified lysate was used at 1:11.85 dilution.
FIGS. 10-18 demonstrate the effect of the mutations on the ability to produce luminescence with and without NLpep.
Example 12
Comparison of Cysteine Vs. Proline as First Amino Acid in Non-Luminescent Peptide
In the following example, a comparison of using cysteine or proline as first amino acid (after necessary methionine) in the non-luminescent peptide was performed. The mutant non-luminescent peptides were generated as previously described in Example 1. Single colony of each non-luminescent polypeptide mutant was grown according to the procedure used in Example 7. The bacterial cultures were also induced according to the procedure used in Example 7. Luminescence was assayed and detected according to the procedure used in Example 7.
FIG. 19 demonstrates that both cysteine and proline can be used as the first amino acid of NLpep and produce luminescence.
Example 13
Identification of the Optimal Composite Set of Mutations for the Non-Luminescent Peptide
In the following examples, an optimal composite set(s) of mutations for the non-luminescent peptide were identified. The mutant non-luminescent peptides were generated as previously described in Example 1.

    
    
        1) For non-luminescent peptide composite mutants NLpep53, NLpep66, NLpep67, and NLpep68, a single colony of each was grown according to the procedure used in Example 10. The bacterial cultures were also induced according to the procedure used in Example 10. TMR gel analysis and luminescence was assayed and detected according to the procedure used in Example 10. The results in FIG. 20 demonstrate the luminescence as well as the E. coli expression of NLpeps containing multiple mutations. 2) For non-luminescent peptide composite mutants NLpep53 and NLpeps 66-74, a single colony of each was grown according to the procedure used in Example 7. The bacterial cultures were also induced according to the procedure used in Example 7. Luminescence was assayed and detected according to the procedure used in Example 7. The results in FIG. 21 demonstrate the luminescence of NLpeps containing multiple mutations.
        3) For non-luminescent peptide
        composite mutants NLpep53 and NLpeps 66-76, a single colony of each was grown according to the procedure used in Example 7. The bacterial cultures were also induced according to the procedure used in Example 7. Luminescence was assayed and detected according to the procedure used in Example 7 except the non-luminescent polypeptide was 5A2 or 5A2+R11E (1:10 dilution of E. coli clarified lysate). The results in FIG. 22 demonstrate the luminescence of NLpeps containing multiple mutations with 5A2 or 5A2+R11E. These results also demonstrate the lower luminescence when the NLpoly mutation R11E is complemented with an NLpep containing E as the 9th residue (NLpep72, 75, and 76).
        4) For non-luminescent peptide composite mutants NLpep1, NLpep69, NLpep78 and NLpep79, a single colony of each was grown according to the procedure used in Example 7. The bacterial cultures were also induced according to the procedure used in Example 7. Luminescence was assayed and detected according to the procedure used in Example 7 except the non-luminescent polypeptide was WT (1:10 dilution of E. coli clarified lysate). The results in FIG. 23 demonstrate the luminescence of NLpeps containing multiple mutations.
    
    


Example 14
Composite Non-Luminescent Polypeptide Mutants
In the following example, 9 mutations from the library screens were combined into a composite clone (NLpoly1, SEQ ID NOS: 941,942), and then one of the mutations reverted back to the original amino acid (NLpoly2-10, SEQ ID NOS: 943-960) in order to identify the optimal composite set. Based on previous results of NLpoly1-10, NLpoly11-13 (SEQ ID NOS: 961-966) were designed and tested for the same purpose. Mutant NLpolys were generated as previously described in Example 1. Single colony of each non-luminescent polypeptide mutant was grown according to the procedure used in Example 6. The bacterial cultures were also induced according to the procedure used in Example 6. Luminescence was assayed and detected according to the procedure used in Example 6 expect NLpep53 E. coli clarified lysate was used at 1:11.85 dilution.
FIG. 24 demonstrates the luminescence of NLpolys containing multiple mutations.
Example 15
Substrate Specificity of Non-Luminescent Polypeptide Mutants
The following example investigates the substrate specificity of the non-luminescent polypeptide mutants. Luminescence generated from luminescent complexes formed from various non-luminescent polypeptide mutants, either Furimazine or coelenterazine as a substrate, and various non-luminescent peptides.
HEK 293 cells were plated at 100,000 cells/ml into wells of a 24 well plates containing 0.5 ml DMEM+10% FBS (50,000/well). The cells were incubated in a 37° C., 5% CO2 incubator overnight. DNA for expression of each non-luminescent polypeptide mutant was transfected in duplicate. 1 ug plasmid DNA containing a non-luminescent polypeptide mutant was mixed with OptiMEM (Life Technologies) to a final volume of 52 ul. 3.3 μl of Fugene HD (Promega Corporation) was added, and samples incubated for 15 minutes at room temperature. 25 μl of each sample mixture was added to two wells and incubated overnight in a 37° C., 5% CO2 incubator overnight. After overnight incubation, the growth media was removed and 0.5 ml DMEM (without phenol red)+0.1% Prionex added. The cells were then frozen on dry ice (for how long) and thawed prior to detecting luminescence.
In FIGS. 25-26, luminescence was assayed and detected according to the procedure used in Example 6, except NLpep53 E. coli clarified lysate was used at 1:10 dilution and either Furimazine or coelenterazine in either NanoGlo Luciferase Assay buffer or DMEM were used. This data demonstrates the luminescence of NLpolys in NANOGLO and DMEM with either Furimazine or Coelenterazine as the substrate. This indicates the substrate specificity (Furimazine versus Coelenterazine) of the NLpoly in both NANOGLO and DMEM.
In FIG. 27, luminescence was assayed and detected according to the procedure used in Example 6, except E. coli clarified lysate from various non-luminescent peptides (NLpep1, NLpep9, NLpep48, NLpep53, NLpep69 or NLpep76) were used at 1:10 dilution. In addition, either Furimazine or coelenterazine in either NanoGlo Luciferase Assay buffer were used. This data demonstrates the substrate specificity of NLpoly/NLpep pairs.
In FIG. 28, luminescence was assayed and detected by separately diluting NLpep53-HT fusion 1:10 and the non-luminescent polypeptide lysates 1:10 in DMEM+0.1% Prionex. 20 μl of non-luminescent peptide and 20 μl non-luminescent polypeptide were then combined and incubated for 10 minutes at room temperature. 40 μl of NanoGlo Buffer with 100 uM Furimazine or DMEM with 0.1% Prionex and 20 uM Furimazine was then added to the samples, and luminescence detected on GloMax Multi. This data demonstrates the substrate specificity of NLpolys expressed in HEK293 cells.
In FIG. 29, luminescence was assayed and detected by separately diluting NLpep1-HT, NLpep53-HT, NLpep69-HT or NLpep76-HT fusion 1:10 and the non-luminescent polypeptide lysates 1:10 in DMEM+0.1% Prionex. 20 μl of non-luminescent peptide and 20 μl non-luminescent polypeptide were then combined and incubated for 10 minutes at room temperature. 40 μl of NanoGlo Buffer with 100 uM Furimazine or DMEM with 0.1% Prionex and 20 uM Furimazine was then added to the samples, and luminescence detected on GloMax Multi. This data demonstrates the luminescence of NLpolys expressed in mammalian cells and assayed with various NLpeps.
Example 16
Signal-to-Background of Non-Luminescent Polypeptide Mutants with Furimazine or Coelenterazine
The following example investigates signal-to-background of the non-luminescent polypeptide mutants. Luminescence generated from various non-luminescent polypeptide mutants was measured using either Furimazine or coelenterazine as a substrate as well as with various non-luminescent peptides.
HEK 293 cells were plated at 15,000 cells/well in 100 μl DMEM+10% FBS into wells of 96-well plates. The cells were incubated in a 37° C., 5% CO2 incubator overnight. Transfection complexes were prepared by adding 0.66 μg each of plasmid DNA for expression of a non-luminescent polypeptide mutant and a non-luminescent peptide mutant plasmid to a final volume of 31 μl in OptiMem. 2 μl Fugene HD was added to each transfection complex and incubated for 15 minutes at room temperature. For each peptide/polypeptide combination, 5 μl of a transfection complex was added to 6 wells of the 96-well plate and grown overnight at 37 C in CO2 incubator. After overnight incubation, the growth media was removed and replaced with CO2— independent media containing either 20 uM coelenterazine or 20 uM Furimazine. The samples were incubated for 10 minutes at 37° C., and kinetics measured over the course of 1 hour at 37° C. on a GloMax Multi+. FIG. 30 demonstrates the substrate specificity of various NLpoly/NLpep pairs when the NLpoly is expressed in mammalian cells.
Example 17
Luminescence and Substrate Specificity
The following example investigates the luminescence and substrate specificity of various non-luminescent polypeptide mutants with NLpep69 and using either Furimazine or coelenterazine as a substrate.
CHO cells were plated at 20,000 cells/well in 100 μl of DMEM+10% FBS into wells of 96-well plates. The cells were incubated in a 37° C., 5% CO2 incubator overnight. Transfection complexes were prepared by adding 0.66 ug each of plasmid DNA for expression of a non-luminescent polypeptide mutant and a non-luminescent peptide mutant plasmid to a final volume of 31 μl in OptiMem. 2 μl Fugene HD was added to each transfection complex and incubated for 15 minutes at room temperature. For each peptide/polypeptide combination, 5 μl of transfection complex was added to 6 wells of the 96-well plate and grown overnight at 37 C in CO2 incubator. After overnight incubation, the growth media was removed and replaced with CO2— independent media containing either 20 uM coelenterazine or 20 uM Furimazine. The samples were incubated for 10 minutes at 37° C., and kinetics measured over the course of 1 hour at 37° C. on a GloMax Multi+. FIG. 31 demonstrates the substrate specificity when NLpolys are coexpressed in mammalian cells with NLpep69.
Example 18
Luminescence and Substrate Specificity Between Live-Cell and Lytic Conditions
The following example investigates the luminescence and substrate specificity of various non-luminescent polypeptide mutants with NLpep69, NLpep78 or NLpep79, using either Furimazine or coelenterazine as a substrate and under either lytic or live cell conditions.
HEK 293 cells were plated at 15,000 cells/well in 100 μl DMEM+10% FBS into wells of 96-well plates. The cells were incubated in a 37° C., 5% CO2 incubator overnight. Transfection complexes were prepared by adding 0.66 ug each of plasmid DNA for expression of a non-luminescent polypeptide mutant and a non-luminescent peptide mutant plasmid to a final volume of 31 μl in OptiMem. 2 μl Fugene HD was added to each transfection complex and incubated for 15 minutes at room temperature. For each NLpoly-NLpep combination, 5 μl of transfection complex was added to 6 wells of the 96-well plate and grown overnight at 37 C in CO2 incubator. After overnight incubation, the growth media was removed and replaced with CO2-independent media containing either 20 uM coelenterazine or 20 uM Furimazine. The samples were incubated for 10 minutes at 37° C., and kinetics measured over the course of 1 hour at 37° C. on a GloMax Multi+. FIGS. 32-34 demonstrate the substrate specificity of NLPolys coexpressed in mammalian cells with NLpep69, 78, or 79 in live-cell and lytic formats.
Example 19
Comparison of Non-Luminescent Polypeptide Mutants Expressed in E. coli 
A single colony of each non-luminescent polypeptide was grown according to the procedure used in Example 7. The bacterial cultures were also induced according to the procedure used in Example 7. Luminescence was assayed and detected according to the procedure used in Example 7 except NLpep78-HT or NLpep79-HT at 1:1,000 dilution was used. FIG. 35 demonstrates the luminescence of NLpolys expressed in E. coli and assayed with NLpep78 or 79.
Example 20
Ability of Non-Luminescent Polypeptide Clones to Produce Luminescence without Complementing Non-Luminescent Peptide
A single colony of each non-luminescent polypeptide was grown according to the procedure used in Example 7. The bacterial cultures were also induced according to the procedure used in Example 7. Luminescence was assayed and detected according to the procedure used in Example 7 except no non-luminescent peptide was added to the assay buffer. FIG. 36 demonstrates the luminescence of NLpolys expressed in E. coli and assayed in the absence of NLpep.
Example 21
Substrate Specificity of Non-Luminescent Polypeptide Mutants Expressed in E. coli 
A single colony of each non-luminescent polypeptide was grown according to the procedure used in Example 7. The bacterial cultures were also induced according to the procedure used in Example 7. Luminescence was assayed and detected according to the procedure used in Example except either Furimazine or coelenterazine was mixed with NANOGLO Assay Buffer. FIG. 37 demonstrates the substrate specificity of NLpolys expressed in E. coli and assayed with NLpep78 or 79.
Example 22
Improved Luminescence of Non-Luminescent Polypeptide Mutants with NLpep78
Complementation of the non-luminescent polypeptide mutants with NLpep78-HT was demonstrated in CHO and Hela cells.
CHO and Hela cells (CHO: 100,000 seeded the day prior to transfection; Hela: 50,000 seeded the day prior to transfection) were transfected with 5 ng of a non-luminescent polypeptide mutant 5A2 or 5P or with wild-type non-luminescent polypeptide using Fugene HD into wells of a 24-well plate and incubated at 37° C. overnight. After the overnight incubation, the media was replaced with DMEM without phenol red, and the cells frozen at −80° C. for 30 minutes. The cells were then thawed and transferred to a 1.5 ml tube. The cell lysates were then diluted 1:10 DMEM without phenol red, 20 μl mixed with NLpep78 (NLpep78-HT7 E. coli lysate diluted 1:1,000 in DMEM without phenol red) and shaken at room temperature for 10 minutes. 40 μl DMEM without phenol red and 20 uM Furimazine were added and luminescence measured on a GloMax with a 0.5 second integration. FIG. 38 demonstrates the luminescence of NLpolys expressed in mammalian cells and assayed with NLpep78.
Example 23
Non-Luminescent Polypeptide Fusions and Normalizing Non-Luminescent Polypeptide Concentrations
A comparison of raw and normalized luminescence from non-luminescent polypeptide fused to either firefly luciferase (FIG. 39) or click beetle red luciferase (FIG. 40) were performed to provide insight into how much benefit, e.g., in expression, solubility and/or stability, stems from the concentration of the non-luminescent polypeptide as well as complementation as a fusion non-luminescent polypeptide.
HEK293, Hela or CHO cells were transfected with 5 ng 5P NLpoly-firefly luciferase fusion, 5P NLpoly-click beetle luciferase fusion, wild-type 5P-firefly luciferase fusion or wild-type 5P-click beetle luciferase fusion according to the procedure in Example 22. Lysates were also prepared according to Example 22. The cell lysates were then diluted 1:10 DMEM without phenol red, 20 μl mixed with NLpep78 (diluted 1:100 in DMEM without phenol red; E. coli lysate) and shaken at room temperature for 10 minutes. 40 μl NanoGlo with 20 uM Furimazine or Bright-Glo (Promega Corporation) was added and luminescence measured on a GloMax with 0.5 second integration. FIGS. 39 and 40 demonstrate the specific activity of 5P versus WT NLpoly expressed in mammalian cells and assayed with NLpep78.
Example 24
Complementation in Live Cells
This example demonstrates complementation in live-cells using either wild-type or 5P NLpoly.
Hela cells plated into wells of 96-well plated, transfected with 0.5 ng of wild-type or 5P non-luminescent polypeptide plasmid DNA using Fugene HD and incubated at 37° C. overnight. After the overnight incubation, the cells were then transfected with 0.5 ng NLpep78-HT plasmid DNA using Fugene HD and incubated at 37° C. for 3 hours. The media was then replaced with CO2-independent media+0.1% FBS and 20 uM PBI-4377 and luminescence measured at 37° C. on a GloMax with 0.5 second integration. FIG. 41 demonstrates the live-cell complementation between 5P or WT NLpoly and NLpep78.
Example 25
Complementation in Cell-Free Extract
To demonstrate complementation in cell-free extract, 0.5 ug NLpep78-HT and 0.5 ug non-luminescent polypeptide mutant plasmid DNA were mixed with TNT rabbit reticulocyte lysate master mix (Promega Corporation) and incubated at 30° C. for 1 hour. 25 μl of the cell-free expression extract was mixed with 25 μl NanoGlo Luciferase Assay reagent and incubated at room temperature for 10 minutes. Luminescence was measured on a GloMax with 0.5 second integration. FIG. 42 demonstrates luminescence from complementing NLpoly/NLpep pairs expressed in a cell-free format.
Example 26
Binding Affinity of Non-Luminescent Polypeptide Expressed in Mammalian Cells with Synthetic Non-Luminescent Peptide
To demonstrate the binding affinity between non-luminescent polypeptide and non-luminescent peptide pairs, non-luminescent polypeptide lysates from Hela, HEK293 and CHO cells were prepared as previously described and diluted 1:10 PBS+0.1% Prionex. 4× concentrations of non-luminescent peptide (synthetic) were made in PBS+0.1% Prionex. 20 μl of the non-luminescent polypeptide lysate was mixed with 20 μl non-luminescent peptide and shaken at room temperature for 10 minutes. 40 μl of NanoGlo Luciferase Assay Reagent or PBS+0.1% Prionex with Furimazine was added and shaken at room temperature for 10 minutes. Luminescence was detected on a GloMax with 0.5 s integration. Kd values were determined using Graphpad Prism, One Site-Specific Binding. FIGS. 43 and 44 demonstrate the dissociation constants measured under various buffer conditions (PBS for complementation then NanoGlo for detection, PBS for complementation and detection, NanoGlo for complementation and detection).
Example 27
Improved Binding Affinity when Cysteine Mutated to Phenylalanine in Non-Luminescent Peptide Mutants
To demonstrate improved binding affinity in non-luminescent peptide mutants with a mutated cysteine at the 8th residue of the peptide, non-luminescent polypeptide mutant lysates from Hela, HEK293 and CHO cells were prepared as previously described and diluted 1:10 PBS+0.1% Prionex. 4× concentrations of non-luminescent peptide (NLpep) were made in PBS+0.1% Prionex+10 mM DTT. 20 μl of the non-luminescent polypeptide lysate was mixed with 20 μl non-luminescent peptide and shaken at room temperature for 10 minutes. 40 μl of NanoGlo Luciferase Assay Reagent was added and shaken at room temperature for 10 minutes. Luminescence was detected on a GloMax with 0.5 s integration. FIG. 45 demonstrates NLpep C8F mutation significantly improves the binding affinity for 5P.
Example 28
Detectable Luminescence of Polypeptide Variants without Non-Luminescent Peptide in Hela Cells
To demonstrate luminescence in non-luminescent polypeptide without non-luminescent peptide, Hela cells (10,000 seeded the day prior to transfection) in wells of a 96-well plate were transfected with varying amounts of non-luminescent polypeptide+pGEM-3zf Carrier DNA to a total of 50 ng using Fugene HD and incubated 37° C. overnight. After incubation, the media was replaced with CO2-independent media+0.1% FBS+20 uM Furimazine and incubated at 37° C. for 10 minutes, and luminescence detected on a GloMax with 0.5 s integration. FIG. 46 demonstrates the luminescence of NLpoly WT or 5P in live Hela cells without NLpep after transfection of various amounts of plasmid DNA.
Example 29
Generation of Additional Non-Luminescent Polypeptide Variants
Additional non-luminescent polypeptide variants: Ile-11 (Ile at residue 11), Val-11, Tyr-11, Glu-11, Glu-157, Pro-157, Asp-157, Ser-157, Met-149, Leu-106, NLpoly11, and NLpoly12 were generated as described below, and their expression analyzed. The additional non-luminescent polypeptide variants were made in the 5A2 non-luminescent polypeptide background.
Fresh individual colonies (KRX) of each additional non-luminescent polypeptide variants were picked and grown overnight in LB+ampicillin (100 ug/ml) at 30° C. and then diluted 1:100 in LB+ampicillin and grown at 37° C. for 2.5 hours (OD600 ̃0.5). Rhamnose was added to a final concentration of 0.2%, and the cells were split in triplicate and grown overnight at 25° C. for  ̃18 h. Cells were lysed using 0.5× Fast Break for 30 minutes at ambient temperature, snap-frozen on dry ice, and stored at −20° C. Upon fast thawing, soluble fractions were prepared by centrifugation at 10K for 15 min at 4° C. Samples were assayed for luminescence on a Tecan Infinite F-500 luminometer.
FIG. 49 demonstrates that total lysate and soluble fraction of each non-luminescent polypeptide variant as analyzed by SDS-PAGE. The data provides information about expression, solubility and stability of the additional non-luminescent polypeptide variants. A majority of the additional non-luminescent polypeptide variants produced more protein (total and soluble) than wild-type, but in many cases, the difference is subtle. Improved expression for NLpoly11 and NLpoly 12 was more noticeable.
Example 30
Background Luminescence of Additional Non-Luminescent Polypeptide Variants
The background luminescence of the additional non-luminescent polypeptide variants generated in Example 29 was measured by incubating 25 μl of non-luminescent polypeptide variant lysate with 25 μl DMEM at room temperature for 10 minutes. 50 μl NanoGlo Luciferase Assay Reagent was then added, and luminescence measured at 5 and 30 minutes on a Tecan Infinite F500. NLpep53 (Pep 53) alone and DMEM (DMEM) alone were used as controls. FIG. 47 demonstrates that a majority of the additional non-luminescent polypeptide variants showed elevated background luminescence.
Example 31
Luminescence of Additional Non-Luminescent Polypeptide Variants after Complementation
Luminescence of the additional non-luminescent polypeptide variants generated in Example 28 was measured by incubating 25 μl of non-luminescent polypeptide variant lysate with 25 μl NLpep-53 at room temperature for 10 minutes 50 μl NanoGlo Luciferase Assay Reagent was then added, and luminescence measured at 5 and 30 minutes on a Tecan Infinite F500. NLpep53 (Pep 53) alone and DMEM (DMEM) alone were used as controls. FIG. 48 demonstrates that the non-luminescent polypeptide variants Val-11, Glu-11, Glu-157, Pro-157, Asp-157, Ser-157 and Met-149 generated significantly more luminescence than parental 5A2.
Example 32
Correlation Between Increased Background Luminescence of Non-Luminescent Polypeptide in the Absence of Non-Luminescent Peptide and Amount of Protein in Soluble Fraction
Individual colonies of the non-luminescent polypeptide variants 3P, 3E, 5P, 5E, 6P and 6E were picked and grown overnight in LB+ampicillin at 30° C. and then diluted 1:100 in LB+ampicillin and grown at 37° C. for 2.5 hours (OD600 ̃0.5). Rhamnose was added to a final concentration of 0.2%, and the cells were split in triplicate and grown overnight at 25° C. for  ̃18 h. Cells were lysed using 0.5× Fast Break for 30 minutes at ambient temperature, snap-frozen on dry ice, and stored at −20° C. Upon fast thawing, soluble fractions were prepared by centrifugation at 10K for 15 min at 4° C. Samples were assayed for luminescence on a Tecan Infinite F-500. FIG. 50A shows the total lysate and soluble fraction of each non-luminescent polypeptide variant. FIG. 50B shows the background luminescence of each non-luminescent polypeptide variant. FIG. 51 shows the luminescence generated with each non-luminescent polypeptide variant when complemented with 10 or 100 nM NLpep78 (NVSGWRLFKKISN) in LB medium.
Example 33
Elongations and Deletions of Non-Luminescent Polypeptide
The non-luminescent polypeptide variant 5P was either elongated at the C-terminus by the addition of the residues VAT, AA, VTG, VT, VTGWR (SEQ ID NO: 2576), VTGW (SEQ ID NO: 2577), V, A, VA, GC; AT, GTA, ATG or GT or deletion of 1 to 7 residues at the C-terminus of 5P, e.g., D1=deletion of 1 residue, D2=deletion of 2 residues, etc. Background luminescence in E. coli lysates (FIG. 52) and luminescence generated after complementation with NLpep78 (FIG. 53; NVSGWRLFKKISN (SEQ ID NO: 374)) or NLpep79 (FIG. 54; NVTGYRLFKKISN(SEQ ID NO: 376)) were measured. FIG. 55 shows the signal-to-background of the non-luminescent polypeptide 5P variants. FIG. 56 provides a summary of the luminescent results. FIG. 57 shows the amount of total lysate and soluble fraction in each non-luminescent polypeptide 5P variant.
Example 34
Comparison of 5P and 1107L Non-Luminescent Polypeptide Variant
FIG. 58 shows the amount of total lysate and soluble fraction of 5P and I107L (A), luminescence generated by 5P or I107L without non-luminescent peptide or with NLpep78 or NLpep79 (B) and the improved signal-to-background of I107L over 5P (C).
Example 35
Generation of 5P Non-Luminescent Polypeptide Mutants
Mutations identified in a screening of random mutations in the 5P non-luminescent polypeptide variant were generated as previously described. Each single 5P non-luminescent polypeptide mutant colony was inoculated in 200 μl Minimal Media and incubated with shaking at 37° C. for 20 hours. 10 μl of the culture was then added to 190 μl of fresh Minimal Media and incubated again with shaking at 37° C. for 20 hours. 10 μl of the second culture was then added to 190 μl Auto-Induction Media (Minimal Media+5% glucose+2% rhamnose) and incubated with shaking at 25° C. for 18 hours to allow expression of the non-luminescent polypeptide mutant. 10 μl of the 5P non-luminescent polypeptide mutant expression culture was added to 40 μl of assay lysis buffer containing NLpep78-HT (1:386 dilution) or NLpep79-HT (1:1,000 dilution) and shaken at room temperature for 10 minutes. 50 μl of NanoGlo Assay Buffer containing 100 uM coelenterazine was added and shaken at room temperature for 10 minutes. Luminescence was measured on GloMax with 0.5 sec integration. FIGS. 59-62A shows background luminescence while FIGS. 59-62B and C show luminescence generated after complementation with NLpep78 or NLpep79.
Example 36
Binding Affinity Between Elongated Non-Luminescent Polypeptide Variant and Deleted Non-Luminescent Peptide
The binding affinity between an elongated non-luminescent polypeptide variant, i.e., containing additional amino acids at the C-terminus, and a deleted non-luminescent peptide, i.e., deleted amino acids at the N-terminus.
Lysates of E. coli expressing non-luminescent polypeptide 5P/+V/+VT/+VTG prepared as previously described were diluted 1:2000 in PBS+0.1% Prionex. 25 μl of the diluted lysate was incubated with 25 μl of NLpep78, NLpep80, NLpep81 or NLpep82 (diluted 0-500 nM in dilution buffer) for 5 min at room temp. 50 μl of Furimazine diluted to 1× with NanoGlo Assay Buffer was added to each sample and incubated for 10 minutes at room temperature. Luminescence was measured on a GloMax Multi with 0.5 s integration time. FIG. 63 demonstrates the binding affinity between NLpolys with additional amino acids at the C-terminus with NLpeps with amino acids deleted from the N-terminus.
Example 37
Binding Affinity Between Non-Luminescent Polypeptide Expressed in E. coli and Synthetic Non-Luminescent Peptide
Non-luminescent polypeptide LB lysates were prepared and diluted 1:100 into PBS+0.1% Prionex. 2× dilutions of synthetic NLpep78 were made in PBS+0.1% Prionex. 25 μl of the diluted non-luminescent polypeptide lysate was mixed with 25 μl of each dilution of non-luminescent peptide and incubated 3 minutes at ambient temperature. 50 μl of NanoGlo Luciferase Assay Reagent was added, incubated for 5 minutes at room temperature, and luminescence measured on a GloMax Multi+. FIG. 64 shows the calculated Kd values using one-site specific binding.
Example 38
Binding Affinity Between 5P Non-Luminescent Polypeptide Expressed in Mammalian Cells and NLpep80 or NLpep87
Lysates of CHO, HEK293T, or HeLa cells expressing NLpoly 5P were diluted 1:1000 in dilution buffer (PBS+0.1% Prionex.) 25 μl of diluted lysate was incubated with 25 μl of NLpep80/87 (diluted 0-5 μM in dilution buffer) for 5 min at room temp. 50 μl of furimazine (diluted to 1× with NanoGlo buffer) was added to each well, and the plate was incubated for 10 min at room temp. Luminescence was then read on a GloMax Multi with 0.5 s integration time (FIG. 65).
Example 39
Binding Affinity Between 5P Non-Luminescent Polypeptide Expressed in E. coli and NLpep80 or NLpep87
Lysates of E. coli expressing NLpoly 5P were diluted 1:2000 in dilution buffer (PBS+0.1% Prionex.) 25 μl of diluted lysate was incubated with 25 μl of NLpep80/87 (diluted 0-5 μM in dilution buffer) for 5 min at room temp. 50 μl of furimazine (diluted to 1× with NanoGlo buffer) was added to each well, and the plate was incubated for 10 min at room temp. Luminescence was then read on a GloMax Multi with 0.5 s integration time (FIG. 66).
Example 40
Complementation Between a Deleted Non-Luminescent Polypeptide and Elongated Non-Luminescent Peptide
Complementation between a deleted non-luminescent polypeptide, i.e., amino acids deleted from the C-terminus, and an elongated non-luminescent peptide, i.e., amino acids added to the N-terminus, was performed. NLpep-HT E. coli clarified lysates as prepared as previously described in Example 6. The amount of NLpep-HT was quantitated via the HaloTag fusion. Briefly, 10 μl of clarified lysate was mixed with 10 μl HaloTag-TMR ligand (diluted 1:100) and 80 μl water and incubated at room temperature for 10 minutes. 33.3 μl 4×SDS Loading Buffer was added and incubated at 95° C. for 5 minutes. 15 μl was loaded onto an SDS-PAGE gel and imaged on a Typhoon. Based on the intensities from the SDS-PAGE gel, non-luminescent peptides were diluted in PBS+0.1% Prionex non-luminescent peptides to make equivalent concentrations. The non-luminescent polypeptide lysates were then diluted 1:100 in PBS+0.1% Prionex. 20 μl of diluted non-luminescent polypeptide and 20 μl diluted non-luminescent peptide were mixed and shaken at room temperature for 10 minutes. 40 μl NanoGlo Luciferase Assay Reagent was added and shaken at room temperature for 10 minutes. Luminescence was measured on a GloMax using 0.5 sec integration. FIG. 67 demonstrates the luminescence of NLpolys with amino acids removed from the C-terminus with NLpeps with additional amino acids on the N-terminus.
Example 41
Binding Affinity Between 5P Non-Luminescent Polypeptide Expressed in Hela Cells and NLpep78 or Truncated NLpep78 (NLpep80-87)
5P non-luminescent polypeptide lysate was prepared from Hela cells as previously described and diluted prepared 1:10 in PBS+0.1% Prionex. 4× concentrations (range determined in preliminary titration experiment) of non-luminescent peptide (synthetic peptide; by Peptide 2.0 (Virginia); made at either 5, 10, or 20 mg scale; blocked at the ends by acetylation and amidation, and verified by net peptide content analysis) was prepared in PBS+0.1% Prionex. 20 μl P non-luminescent polypeptide and 20 μl non-luminescent peptide were mixed and shaken at room temperature for 10 minutes. 40 μl of NanoGlo Luciferase Assay reagent was added and shaken at room temperature for 10 minutes. Luminescence was measured on GloMax with 0.5 s integration. FIG. 68 demonstrates the binding affinity and corresponding luminescence between 5P and truncated versions of NLpep78. The binding affinity is increased when 1 amino acid is removed from the N-terminus, the C-terminus, or 1 amino acid from each terminus. Removing more than 1 amino acid from either terminus lowers the affinity but does not always lower the Vmax to the same extent.
Example 42
Binding Affinity Between Elongated Non-Luminescent Polypeptide and Truncated Non-Luminescent Peptide
The binding affinity between an elongated non-luminescent polypeptide, i.e., one with 2 extra amino acids on C-terminus, and a truncated non-luminescent peptide, i.e., one with 2 amino acids removed from N-terminus (NLpep81), was determined.
Non-luminescent polypeptide lysate was prepared as previously described and diluted prepared 1:100 in PBS+0.1% Prionex. 2× dilutions of NLpep81 (synthetic peptide; by Peptide 2.0 (Virginia); made at either 5, 10, or 20 mg scale; blocked at the ends by acetylation and amidation, and verified by net peptide content analysis) was prepared in PBS+0.1% Prionex. 25 μl non-luminescent polypeptide and 25 μl of each non-luminescent peptide dilution were mixed and shaken at room temperature for 3 minutes. 50 μl of NanoGlo Luciferase Assay reagent was added and shaken at room temperature for 5 minutes. Luminescence was measured on GloMax with 0.5 s integration. FIG. 69 shows the calculate Kd values using one-site specific binding.
Example 43
Binding Affinity Between Elongated Non-Luminescent Polypeptide and Truncated Non-Luminescent Peptide
The binding affinity between an elongated non-luminescent polypeptide, i.e., one with 3 extra amino acids on C-terminus, and a truncated non-luminescent peptide, i.e., one with 3 amino acids removed from N-terminus (NLpep82), was determined.
Non-luminescent polypeptide lysate was prepared and diluted prepared 1:100 in PBS+0.1% Prionex. 2× dilutions of NLpep82 (synthetic peptide; by Peptide 2.0 (Virginia); made at either 5, 10, or 20 mg scale; blocked at the ends by acetylation and amidation, and verified by net peptide content analysis) was prepared in PBS+0.1% Prionex. 25 μl non-luminescent polypeptide and 25 μl of each non-luminescent peptide dilution were mixed and shaken at room temperature for 3 minutes. 50 μl of NanoGlo Luciferase Assay reagent was added and shaken at room temperature for 5 minutes. Luminescence was measured on GloMax with 0.5 s integration. FIG. 70 shows the calculate Kd values derived using one-site specific binding.
Example 44
Binding Affinity Between Non-Luminescent Polypeptide Clones Expressed in E. coli and Synthetic NLpep78
Non-luminescent polypeptide variants were grown in M9 minimal media. Individual colonies were inoculated and grown overnight at 37° C. Samples were diluted 1:20 in M9 minimal media and grown overnight at 37° C. Samples were again diluted 1:20 in M9 induction media and grown overnight at 25° C. Samples were pooled, and 100 μl of the pooled cells were lysed with 400 μl of PLB lysis buffer and incubate at room temperature for 10 minutes. The lysates were diluted 1:100 in PBS+0.1% Prionex. 2× dilutions of synthetic NLpep78 were made in PBS+0.1% Prionex. 25 μl of non-luminescent polypeptide dilution was mixed with 25 μl of each non-luminescent peptide dilution and incubated for 3 minutes at room temperature. 50 μl of NanoGlo Luciferase Assay Reagent was added, incubated at room temperature for 5 minutes, and luminescence read on GloMax Multi+. FIG. 71 shows the calculate Kd values derived using one-site specific binding.
Example 45
Determination of the Effect of Mutations on Km
Using diluted pooled lysates from Example 11, 25 μl of non-luminescent polypeptide diluted lysate (1:100 in PBS+0.1% Prionex) was mixed with 25 μl of 500 nM NLpep78 for each sample and incubated at room temperature for 5 minutes. 2× dilutions of Furimazine in NanoGlo Luciferase Assay Buffer were prepared, and 50 μl of non-luminescent peptide and non-luminescent polypeptide sample mixed with 50 μl of NanoGlo/Furimazine dilutions. Luminescence was measured after 5 minute incubation at room temperature. FIG. 72 show the calculated Km derived using Michaelis-Menten.
Example 46
Demonstration of a Three-Component Complementation
A tertiary complementation using 2 NLpeps and NLpoly 5P non-luminescent polypeptide is demonstrated. NLpoly 5P-B9 (5P with residues 147-157 deleted) and NLpep B9-HT (Met+ residues 147-157 fused to N-terminus of HT7) lysates were prepared.
A) NLpoly 5P-B9+NLpoly B9 Titration with NLpep78
NLpoly 5P-B9+NLpoly B9 was titrated with NLpep78. 20 μl 5P-B9 (undiluted) was mixed with 20 μl peptideB9-HT (undiluted). Dilutions of NLpep78 (synthetic peptide, highest concentration=100 uM) were made in PBS+0.1% Prionex. 20 μl NLpep78 was added to 40 μl of the 5P-B9+ peptideB9-HT mixture and shaken at room temperature for 10 minutes. 60 μl NanoGlo Luciferase Assay Reagent was added and shaken at room temperature for 10 minutes. Luminescence was measured on GloMax with 0.5 s integration.
B) NLpoly 5P-B9+NLpep78 Titration with NLpepB9-HT
20 μl NLpoly 5P-B9 (undiluted) was mixed with 20 μl NLpep78 (100 uM). Dilutions of peptideB9-HT (highest concentration=undiluted) were made in PBS+0.1% Prionex. 20 μl of peptideB9-HT was added to 40 μl of the 5P-B9+NLpep78 mixture and shaken at room temperature for 10 minutes. 60 μl NanoGlo Luciferase Assay Reagent was added and shaken at room temperature for 10 minutes. Luminescence was measured on GloMax with 0.5 s integration.
FIG. 73 demonstrates the feasibility of a ternary system consisting of 2 different NLpeps and a truncated NLpoly. Since all 3 components are non-luminescent without the other 2, this system could be configured such that each NLpep is fused (synthetically or genetic engineering) to a binding moiety and the truncated NLpoly used at high concentrations to produce light only in the presence of an interaction between the binding moieties, or such that each of the 3 components are fused to binding moieties to produce light only in the event of ternary complex formation.
Example 47
Complementation with NLpep88 (NLpep78 with Gly as 6th Residue Instead of Arg)
NLpep88-HT and 5P E. coli clarified lysates were prepared as previously described. Serial dilutions of NLpep88-HT lysate were made in PBS+0.1% Prionex. 20 μl of 5P lysate and 20 μl NLpep88-HT lysate were mixed and shaken at room temperature for 10 minutes. 40 μl of NanoGlo Luciferase Assay Reagent was added and shaken at room temperature for 10 minutes. Luminescence was measured on GloMax with 0.5 s integration. FIG. 74 demonstrates the importance of the arginine residue at the 6th position of the NLpep. While there is no increase in luminescence above 5P alone at lower concentrations of NLpep88, high concentrations of NLpep increased the luminescence suggesting a catalytically compromised complex and not a lack of interaction between 5P and NLpep88.
Example 48
Subcellular Localization of NLpep78 and 79 as N-Terminal Fusions to HaloTag
U2OS cells were plated and left to recover overnight at 37° C. Cells were then transfected with HaloTag alone DNA construct or the HaloTag-NanoLuc peptide DNA constructs (all under the control of CMV promoter): P1-HT, P78-HT or P79-HT diluted 1:10 with carrier DNA (pSI) using FuGENE HD and incubated for 24 hours at 37° C. Cells were then labeled with HaloTag-TMR ligand by the manufacturer's standard rapid labeling protocol and imaged. FIG. 75 demonstrates that NLpep78 and 79 do not alter the intracellular localization of the HaloTag protein.
Example 49
Subcellular Localization of Non-Luminescent Polypeptide (WT and 5P)
U2OS cells were plated and left to recover overnight at 37° C. Cells were either kept as non-transfection controls or transfected with the NanoLuc DNA constructs: FL, NLpoly (wt) or NLpoly(5P) diluted 1:10 with carrier DNA (pSI) using FuGENE HD and incubated for 24 hours at room temperature. Cells were fixed and subsequently processed for ICC. ICC was done using 1:5000 GS (PRO) primary antibody overnight at 4° C. followed by an Alexa488 goat anti-rabbit secondary antibody. FIG. 76 demonstrates that both NLpoly WT and NLpoly 5P localize uniformly in cells.
Example 50
Demonstration that Non-Luminescent Polypeptide can Easily and Quickly Detect Non-Luminescent Peptide Conjugated to a Protein of Interest
99 μl of NLpep53-HT E. coli clarified lysate was mixed with 24.75 μl 4×SDS loading buffer. 1:10 serial dilutions of the lysate-loading buffer mixture were made and incubated at 95° C. for 5 minutes. 15 μl was loaded onto a SDS-PAGE gel. After gel completions, it was transferred to PVDF using iBlot and washed with 10 mL NLpoly L149M E. coli clarified lysate at room temperature for 30 minutes. The membrane was then placed on a LAS4000 imager and 2 mL NanoGlo® Luciferase Assay Reagent added. A 60 second exposure was taken (FIG. 77).
Example 51
Site Saturation at Non-Luminescent Polypeptide Positions 31, 46, 108, 144, and 157 in the Context of 5P
Single amino acid change variants were constructed onto NLpoly 5P (pF4Ag vector background) at the sites according to table 5 below. In effect, the native residue was varied to each of the 19 alternative amino acids for a total of 95 variants.











TABLE 5





Position 31
Position 46
Position 108
Position 144
Position 157
























B1
Ala
E3
Ala
H5
Ala
C8
Ala
F10
Ala


C1
Cys
F3
Cys
A6
Cys
D8 
Cys
G10
Cys


D1
Asp
G3
Asp
B6
Asp
E8
Asp
H10
Asp


E1
Glu
H3
Glu
C6
Glu
F8
Glu
A11
Glu


F1
Gly
A4
Phe
D6
Phe
G8
Phe
B11
Phe


G1
His
B4
Gly
E6
Gly
H8
Gly
C11
Gly


H1
Ile
C4
His
F6
His
A9
His
D11
His


A2
Lys
D4
Ile
G6
Ile
B9
Ile
E11
Ile


B2
Leu
E4
Lys
H6
Lys
C9
Lys
F11
Lys


C2
Met
F4
Met
A7
Leu
D9
Leu
G11
Leu


D2
Asn
G4
Asn
B7
Met
E9
Met
H11
Met


E2
Pro
H4
Pro
C7
Pro
F9
Asn
A12
Asn


F2
Gln
A5
Gln
D7
Gln
G9
Pro
B12
Gln


G2
Arg
B5
Arg
E7
Arg
H9
Gln
C12
Arg


H2
Ser
C5
Ser
F7
Ser
A10
Arg
D12
Ser


A3
Thr
D5 
Thr
G7
Thr
B10
Ser
E12
Thr


B3
Val
E5
Val
H7
Val
C10
Val
F12
Val


C3
Trp
F5
Trp
A8
Trp
D10
Trp
G12 
Trp


D3
Tyr
G5
Tyr
B8
Tyr
E10
Tyr
H12 
Tyr









Individual colonies were grown in LB+ amp and incubated overnight at 30° C. A 5P control was also included. The overnight cultures were used to inoculate fresh LB+ amp (1:100), and these cultures grew for 2 hours 45 minutes at 37° C. Rhamnose was added to 0.2%, and the cultures left to grow/induce overnight at 25° C. After 18 hours of induction, cells were lysed using 0.5× FastBreak (30 min ambient temperature), snap frozen on dry ice, and stored at −20° C. Following a fast thaw, samples were assayed in the absence and presence of Pep87 (aka NLpep 87).
For the (−) peptide reactions, 30 uL lysate was incubated with 30 uL PBS pH 7.5 for 10 min and then 60 uL NanoGlo® Luciferase Assay reagent (Promega Corporation) added. After 5 minutes, luminescence was measured. For the (+) peptide reactions, 30 uL lysate was incubated with 30 uL of 8 nM Pep87. After 10 min, 60 uL NanoGlo® Luciferase Assay reagent was added, and luminescence measured at 5 minutes.
Luminescence (RLU) data for the (−) peptide samples were normalized to the readings for the 5P control, and these results are presented in FIG. 78. Luminescence (RLU) data for the (+) peptide samples were also normalized to 5P, but then also normalized to the values in FIG. 76 in order to represent signal to background (S/B; FIG. 79).
Example 52
Use of the High Affinity Between NLpoly and NLpep for Protein Purification/Pull Downs
MAGNEHALOTAG beads (Promega Corporation; G728A) were equilibrated as follows:
a) 1 mL of beads were placed on magnet for  ̃30 sec, and the buffer removed; b) the beads were removed from magnet, resuspended in 1 mL PBS+0.1% Prionex, and shaken for 5 min at RT; and c) steps a) and b) were repeated two more times NLpep78-HaloTag (E. coli clarified lysate) was bound to MAGNEHALOTAG beads by resuspending the beads in 1 mL NLpep78-HT clarified lysate, shaking for 1 hr at RT and placing on magnet for  ̃30 sec. The lysate (flow through) was removed and saved for analysis. NLpoly 8S (E. coli clarified lysate) was bound to the NLpep78 bound-MagneHaloTag beads from the step above by resuspending the beads in 1.5 mL 8S lysate, shaking for 1 hr at RT and placing on a magnet for  ̃30 sec. The lysate (flow through) was removed and saved for analysis. The beads were resuspended in 1 mL PBS+0.1% Prionex, shaken for 5 min at RT, placed on magnet for  ̃30 sec, and PBS (wash) removed. The beads were washed three more times.
To elute the bound peptide/polypeptide, the beads were resuspended in 500 uL 1×SDS buffer and shaken for 5 min at RT. The beads were then placed on a magnet for  ̃30 sec; the SDS buffer (elution) removed and saved for analysis. The elution was repeated one more time.
The samples were then analyzed by gel. 37.5 uL of sample (except elutions) was mixed with 12.5 uL 4×SDS buffer and incubated at 95° C. for 5 min. 5 uL was loaded onto a Novex 4-20% Tris-Glycine gel and run at −180V for −50 min. The gel was stained with SimplyBlue Safe Stain and imaged on a LAS4000 imager.
FIG. 94 illustrates that the affinity of NLpoly and NLpep is sufficient to allow for purification from an E. coli lysate. As NLpoly 8S was purified from an E. coli lysate, it is reasonable to expect a protein fused to NLpoly 8S (or other variant described herein) could also be purified in a similar fashion. While in this example the NLpep was immobilized and used to purify NLpoly, it is also reasonable to expect a similar result if NLpoly were immobilized.
Example 53
Kinetics of NLpoly/NLpep Binding
2× concentrations of synthetic NLpep were made and diluted 2.7-fold nine times (10 concentrations) in PBS+0.1% Prionex. Final concentrations used in the assay were 30 uM-3.9 nM. WT NLpoly (E. coli clarified lysate; 1:10,000) or 11S (1:10,000,000) was diluted in NanoGlo+100 uM Furmazine (Fz). 50 uL of NLpep was placed into wells of white 96-well assay plate. 50 uL NLpoly/NanoGlo/Fz was injected into the wells using the injector on GloMax® Multi+ instrument, and luminescence measured every 3 sec over 5 min. kobs was found by fitting data to: Y=Ymax(1−e−kobst) using Graphpad Prism. kon and koff were then fitted to: kobs=[NLpep]kon+koff. FIG. 95 illustrates the association and dissociation rate constants for the binding between NLpolys and NLpeps.
Example 54
NLpoly/NLpep Substrate Affinity
NLpoly was diluted into PBS+0.1% Prionex as follows: WT at 1:105, 5P at 1:107, and 11S at 1:108. NLpep was diluted into PBS+0.1% Prionex as follows: 30 uM for WT NLpoly studies or 3 uM for NLpoly 5P and 11S studies. 50 uL NLpoly/NLpep was incubated at RT for 5 min, 50 uL NanoGlo+Fz (ranging from 100 uM to 1.2 uM, 2×) added, and incubated for 10 min at RT. Luminescence was measured on GloMax® Multi+ with 0.5 sec integration. Km was derived using Graphpad Prism, Michaelis-Menton best-fit values. FIG. 96 illustrates the Km values for various NLpoly/NLpep pairs.
Example 55
Substrate Effect on NLpoly/NLpep Affinity
11S (E. coli clarified lysate) was diluted into PBS+0.1% Prionex at 1:107. Synthetic NLpep79 was diluted serially (1:2) from 800 nM to 0.39 nM (2×). 20 uL 11S+20 uL NLpep79 were then mixed and incubated for 5 min at RT. 40 uL NanoGlo+5 uM or 50 uM Fz was added and incubated another 5 min at RT. Luminescence was measured on GloMax® Multi+ with 0.5 sec integration. Kd was derived using Graphpad prism, One site-Specific binding value. FIG. 97 illustrates that saturating concentrations of furimazine increase the affinity between 11S and NLpep79.
Example 56
Km for NLpoly 5A2:NLpep
NLpoly 5A2 was diluted into PBS+0.1% Prionex at 1:105. NLpep (WT, NLpep 78 or NLpep79) was diluted into PBS+0.1% Prionex to 30 uM. 50 uL NLpoly/NLpep was incubated at RT for 5 min. 50 uL NanoGlo+Fz (ranging from 100 uM to 1.2 uM, 2×) was added and incubated for 10 min at RT. Luminescence was measured on GloMax® Multi+ with 0.5 sec integration. Km was derived using Graphpad Prism, Michaelis-Menton best-fit values. FIG. 98 illustrates the Km values for NLpoly5A2 and NLpep WT, 78, and 79.
Example 57
Luminescence of NLpoly without NLpep
E. coli clarified lysate were prepared as described previously for NLpoly WT, 5A2, 5P, 8S and 11S. 50 uL of each lysate and 50 uL NanoGlo+Fz were mixed and incubated for 5 min RT. Luminescence was measured on GloMax® Multi+ with 0.5 sec integration. FIG. 99 illustrates that the ability of the NLpoly to produce luminescence in the absence of NLpep gradually increased throughout the evolution process resulting in  ̃500 fold higher luminescence for 11S than WT NLpoly.
Example 58
Improved Luminescence in E. coli Throughout Evolution Process
A single NLpoly colony of WT, 5A2, 5P, 8S or 11S was inoculated in 200 uL minimal media and grown for 20 hrs at 37° C. on shaker. 10 uL of the overnight culture was diluted into 190 uL fresh minimal media and grown for 20 hrs at 37° C. on shaker. 10 uL of this overnight culture was diluted into 190 uL auto-induction media (previously described) and grown for 18 hrs at 25° C. on shaker. The auto-induced cultures were diluted 50-fold (4 uL into 196 uL assay lysis buffer), 10 uL expression culture added to 40 uL of assay lysis buffer containing NLpep (synthetic; 1 nM; WT, NLpep78, NL79 or NLpep80) and shaken for 10 min at RT. 50 uL NanoGlo+Fz was added, and samples shaken for 5 min at RT. Luminescence was measured on a GloMax luminometer with 0.5 sec integration. FIG. 100 illustrates the improvement in luminescence from E. coli-derived NLpoly over the course of the evolution process, an overall  ̃105 improvement (from NLpolyWT:NLpepWT to NLpoly11S:NLpep80).
Example 59
Improved Luminescence in HeLa Cells Throughout Evolution Process
50 ng plasmid DNA expressing NLpoly WT, 5A2, 5P, 8S or 11S was transfected into HeLa cells into wells of a 12-well plate using FugeneHD. The cells were then incubated overnight at 37° C./5% CO2. The media was replaced with 500 uL DMEM without phenol red, and the cells frozen at −80° C. for >30 min. The cells were thawed and transferred to 1.5 mL tubes.
NLpep WT, NLpep78, NLpep79 or NLpep 80 (synthetic) were diluted to 10 nM in PBS+0.1% Prionex, and 25 ul mixed with 25 uL of each of the NLpoly cell lysate. The samples were shaken for 10 min at RT, and then 50 uL NanoGlo+100 uM Fz added and incubated for 5 min at RT. Luminescence was measured on a GloMax luminometer with 0.5 s integration. FIG. 101 illustrates the improvement in luminescence from HeLa-expressed NLpoly over the course of the evolution process, an overall  ̃105 improvement (from NLpolyWT:NLpepWT to NLpoly11S:NLpep80).
Example 60
Improved Luminescence in HEK293 Cells Throughout Evolution Process
50 ng plasmid DNA expressing NLpoly WT, 5A2, 5P, 8S or 11S was transfected into HEK293 cells into wells of a 12-well plate using FugeneHD. The cells were then incubated overnight at 37° C./5% CO2. The media was replaced with 500 uL DMEM without phenol red, and the cells frozen at −80° C. for >30 min. The cells were thawed and transferred to 1.5 mL tubes.
NLpep WT, NLpep78, NLpep79 or NLpep 80 (synthetic) were diluted to 10 nM in PBS+0.1% Prionex, and 25 ul mixed with 25 uL of each of the NLpoly cell lysate. The samples were shaken for 10 min at RT, and then 50 uL NanoGlo+100 uM Fz added and incubated for 5 min at RT. Luminescence was measured on a GloMax luminometer with 0.5 s integration. FIG. 102 illustrates the improvement in luminescence from HEK293-expressed NLpoly over the course of the evolution process, an overall  ̃104 improvement (from NLpolyWT:NLpepWT to NLpoly11S:NLpep80).
Example 61
Improved Binding Affinity Throughout Evolution
NLpoly WT, 5A2, 5P, 8S or 11S (E. coli clarified lysates) were diluted into PBS+0.1% Prionex as follows: WT 1:104; 5A2 1:105; 5P 1:106; 8S 1:107; and 11S 1:107. NLpepWT, NLpep78, NLpep79 or NLpep80 (synthetic) were serially into PBS+0.1% Prionex to 4× concentration. 25 uL NLpoly and 25 uL NLpep were mixed and incubated for 10 min at RT. 50 uL NanoGlo+100 uM Fz was added and incubated for 5 min at RT. Luminescence was measured on a GloMax Multi+ with 0.5 sec integration. Kd was determined using Graphpad Prism, One Site-Specific Binding, Best-fit values. FIG. 103 illustrates a 104 fold improved affinity (starting affinity: NLpolyWT:NLpepWT, Kd ̃10 uM) of Kd<1 nM (NLpoly11S:NLpep86 or NLpoly11S:NLpep80) of the variants tested over wild-type.
Example 62
NLpoly Luminescence
Single NLpoly variant colonies were inoculated with 200 uL minimal media and grown for 20 hrs at 37° C. on a shaker. 10 uL of the overnight culture were diluted into 190 uL fresh minimal media and grown for 20 hrs at 37° C. on a shaker. 10 uL of this overnight culture was then diluted into 190 uL auto-induction media (previously described) and grown for 18 hrs at 25° C. on a shaker. 10 uL of this expression culture was mixed with 40 uL of assay lysis buffer (previously described) without NLpep or NLpep78-HT (1:3,860 dilution) or NLpep79-HT (1:10,000 dilution) and shaken for 10 min at RT. 50 uL of NanoGlo+Fz was added and again shake for 10 min at RT. Luminescence was measured on GloMax® luminometer with 0.5 sec integration. FIGS. 105-107 illustrate the luminescence of various NLpolys in the absence of NLpep.
Example 63
Solubility of NLpoly Variants
A single NLpoly variant colony (SEE FIG. 143) was inoculated into 5 mL LB culture and incubated at 37° C. overnight with shaking. The overnight culture was diluted 1:100 into fresh LB and incubated at 37° C. for 3 hrs with shaking. Rhamnose was added to the cultures to 0.2% and incubated 25° C. overnight with shaking. 900 ul of these overnight cultures were mixed with 100 uL 10× FastBreak Lysis Buffer (Promega Corporation) and incubated for 15 min at RT. A 75 uL aliquot (total) was removed from each culture and saved for analysis. The remaining culture from each sample were centrifuged at 14,000×rpm in a benchtop microcentrifuge at 4° C. for 15 min. A 75 uL aliquot of supernatant (soluble) was removed from each sample and saved for analysis. 25 uL of 4×SDS buffer was added to the saved aliquots and incubated at 95° C. for 5 min. 5 ul of each sample was loaded onto a 4-20% Tris-Glycine SDS gel and run at  ̃190V for  ̃50 min. The gel was stained with SimplyBlue Safe Stain and imaged on a LAS4000.
FIG. 143 shows a protein gel of total lysates and the soluble fraction of the same lysate for the NLpoly variants.
Example 64
Dissociation Constants
NLpoly variant lysate (SEE FIG. 144; prepared as described previously) was diluted 1:10 into PBS+0.1% Prionex. 4× concentrations of NLpep78 (synthetic NLpep78) were made in PBS+0.1% Prionex. 20 uL NLpoly variant lysate and 20 uL NLpep were mixed and shaken for 10 min at RT. 40 uL NanoGlo/Fz was added and shaken for 10 min at RT. Luminescence was measured on a GloMax® luminometer with 0.5 s integration. Kd determined using Graphpad Prism, One site-specific binding, best-fit values. FIG. 144 illustrates dissociation constants of NLpep78 with various NLpolys.
Example 65
Comparison of Luminescence Generated by Cells Expressing Different Combinations of FRB and FKBP Fused to NLpolySP and NLpep80/87
HEK293T cells (400,000) were reverse-transfected with 1 μg pF4A Ag FKBP or 1 μg pF4A Ag FRB vectors expressing N- or C-terminal fusions of NLpolySP and/or NLpep80/87 using FuGENE HD at a DNA-to-FuGENE HD ratio of 1:4. 24-hours post transfection, cells were trypsinized and re-plated in opaque 96-well assay plates at a density of 10,000 cells per well. 24-hours after plating, cells were washed with PBS and then incubated with or without 20 nM rapamycin for 15, 60 or 120 min in phenol red-free OptiMEMI. 10 μM furimazine substrate with or without 20 nM rapamycin in OptiMEM was added directly to each well and incubated at room temperature for 5 min. Luminescence was then measured on a GloMax Multi with 0.5 s integration time. FIG. 108 (15 min induction), 109 (60 min induction) and 110 (120 min induction) illustrate a general increase in induction over time, with NLpolySP and NLpep80 combinations generating the most luminescence. Individual components contribute minimally to signal.
Example 66
Comparison of Luminescence Generated by Cells Expressing Different Combinations of FRB and FKBP Fused to NLpolySP and NLpep80/87
Although similar to Example 65, this example tested all 8 possible combinations of FRB and FKBP fused to NLpoly/NLpep as well as used less total DNA. HEK293T cells (400,000) were reverse-transfected with a total of 0.001 μg pF4A Ag FRB-NLpolySP and 0.001 μg pF4A Ag FKBP-NLpep80/NLpep87 using FuGENE HD at a DNA-to-FuGENE ratio of 1 to 8. pGEM-3Zf(+) DNA was added to bring total DNA in each transfection to 1 μg. 24-hours post-transfection, 10,000 cells were re-plated in opaque 96-well assay plates and incubated an additional 24 hours. Cells were washed with PBS and then incubated in phenol red-free OptiMEMI with 0 or 50 nM rapamycin for 2 h. 10 μM furimazine substrate (final concentration on cells) with 0 or 50 nM rapamycin in OptiMEM was added directly to each well and incubated at room temperature for 5 min. Luminescence was then measured on a GloMax Multi with 0.5 s integration time. FIG. 111 illustrates that NLpep80 combinations generated the highest luminescence and that all configurations respond to rapamycin treatment.
Example 67
Comparison of Luminescence Generated by FRB or FKBP Fusions Expressed in the Absence of Binding Partner
HEK293T cells (400,000) were reverse-transfected with a total of 0.001 μg pF4A Ag FRB-NLpolySP or pF4A Ag FKBP-NLpep80/NLpep87 using FuGENE HD at a DNA-to-FuGENE ratio of 1 to 8. pGEM-3Zf(+) DNA was added to bring total DNA in each transfection to 1 μg. 24-hours post-transfection, 10,000 cells were re-plated in opaque 96-well assay plates and incubated an additional 24 hours. Cells were washed with PBS and then incubated in phenol red-free OptiMEMI with 0 or 50 nM rapamycin for 2 h. 10 μM furimazine substrate (final concentration on cells) with 0 or 50 nM rapamycin in OptiMEM was added directly to each well and incubated at room temperature for 5 min. Luminescence was then measured on a GloMax Multi with 0.5 s integration time. FIG. 112 illustrates that the individual components generate a low basal level of luminescence that is not responsive to rapamycin treatment.
Example 68
Comparison of Luminescence Generated by Cells Transfected with Varying Amounts of FRB-NLpolySP and FKBP-NLpep80/87 DNA
HEK293T (400,000) cells were reverse-transfected with a total of 2, 0.2, 0.02, or 0.002 μg pF4A Ag FRB-NLpolySP and pF4A Ag FKBP-NLpep80 using FuGENE HD at a DNA-to-FuGENE ratio of 1 to 4. pGEM-3Zf(+) DNA was added to bring total DNA in each transfection to 2 μg. 24-hours post-transfection, 10,000 cells were re-plated in opaque 96-well assay plates and incubated an additional 24 hours. Cells were washed with PBS and then incubated in phenol red-free OptiMEMI with or without 20 nM rapamycin for 2 h. 10 μM furimazine substrate (final concentration on cells) with or without 20 nM rapamycin in OptiMEM was added directly to each well and incubated at room temperature for 5 min. Luminescence was then measured on a GloMax Multi with 0.5 s integration time. FIG. 113 illustrates that transfection with less DNA decreases overall luminescence but increases fold induction.
Example 69
Comparison of Luminescence Generated by Cells Transfected with Varying Amounts of FRB-NLpolySP or FKBP-NLpep80/87 DNA in the Absence of Binding Partner
HEK293T cells (400,000) were reverse-transfected with a total of 2, 0.2, 0.02, or 0.002 μg pF4A Ag FRB-NLpolySP or pF4A Ag FKBP-NLpep80 using FuGENE HD at a DNA-to-FuGENE ratio of 1 to 4. pGEM-3Zf(+) DNA was added to bring total DNA in each transfection to 2 μg. 24-hours post-transfection, 10,000 cells were replated in opaque 96-well assay plates and incubated an additional 24 hours. Cells were washed with PBS and then incubated in phenol red-free OptiMEMI with or without 20 nM rapamycin for 2 h. 10 μM furimazine substrate (final concentration on cells) with or without 20 nM rapamycin in OptiMEM was added directly to each well and incubated at room temperature for 5 min. Luminescence was then measured on a GloMax Multi with 0.5 s integration time. FIG. 114 illustrates that lower DNA levels do not change overall luminescence of cells transfected with individual components.
Example 70
Comparison of Luminescence Generated by Cells Transfected with Varying Amounts of FRB-NLpolySP and FKBP-NLpep80/87 DNA
HEK293T cells (400,000) were reverse-transfected with a total of 0.2, 0.02, 0.002, or 0.0002 μg pF4A Ag FRB-NLpolySP and pF4A Ag FKBP-NLpep80/NLpep87 using FuGENE HD at a DNA-to-FuGENE ratio of 1 to 4. pGEM-3Zf(+) DNA was added to bring total DNA in each transfection to 2 μg. 24-hours post-transfection, 10,000 cells were re-plated in opaque 96-well assay plates and incubated an additional 24 hours. Cells were washed with PBS and then incubated in phenol red-free OptiMEMI with or without 50 nM rapamycin for 2 h. 10 μM furimazine substrate (final concentration on cells) with or without 50 nM rapamycin in OptiMEM was added directly to each well and incubated at room temperature for 5 min. Luminescence was then measured on a GloMax Multi with 0.5 s integration time. FIG. 115 illustrates that luminescence above background, as determined in Examples 69 and 71, and rapamycin induction can be achieved with DNA levels down to 2.5 pg.
Example 71
Comparison of Luminescence Generated by Cells Transfected with Varying Amounts of FRB-NLpolySP or FKBP-NLpep80/87 DNA in the Absence of Binding Partner
HEK293T cells (400,000) were reverse-transfected with a total of 0.2, 0.02, 0.002, or 0.0002 μg pF4A Ag FRB-NLpolySP or pF4A Ag FKBP-NLpep80/NLpep87 using FuGENE HD at a DNA-to-FuGENE ratio of 1 to 4. pGEM-3Zf(+) DNA was added to bring total DNA in each transfection to 2 μg. 24-hours post-transfection, 10,000 cells were re-plated in opaque 96-well assay plates and incubated an additional 24 hours. Cells were washed with PBS and then incubated in phenol red-free OptiMEMI with or without 50 nM rapamycin for 2 h. 10 μM furimazine substrate (final concentration on cells) with or without 50 nM rapamycin in OptiMEM was added directly to each well and incubated at room temperature for 5 min. Luminescence was then measured on a GloMax Multi with 0.5 s integration time. FIG. 116 illustrates no significant change in luminescence generated by individual components when less DNA was used.
Example 72
Comparison of Luminescence Generated by Cells Transfected with Varying Amounts of FRB-NLpolySP and FKBP-NLpep80 or FKBP-NLpep87 DNA after Treatment with Rapamycin for Different Lengths of Time
HEK293T cells (400,000) were reverse-transfected with a total of 2, 0.2, 0.02, or 0.002 μg pF4A Ag FRB-NLpolySP and pF4A Ag FKBP-NLpep80 or FKBP-NLpep87 using FuGENE HD at a DNA-to-FuGENE ratio of 1 to 4. pGEM-3Zf(+) DNA was added to bring total DNA in each transfection to 2 μg. 24-hours post-transfection, 10,000 cells were re-plated in opaque 96-well assay plates and incubated an additional 24 hours. Cells were washed with PBS and then incubated in phenol red-free OptiMEMI with or without 20 nM rapamycin for 5/15/30/60/120 min. 10 μM furimazine substrate (final concentration on cells) with or without 20 nM rapamycin in OptiMEM was added directly to each well and incubated at room temperature for 5 min. Luminescence was then measured on a GloMax Multi with 0.5 s integration time. FIGS. 117 and 118 illustrates a decline in luminescence with less DNA and an increase in rapamycin induction over time.
Example 73
Comparison of Luminescence Generated by Cells Expressing Different Combinations of FRB-NLpolySP or FRB-NLpoly5A2 with FKBP-NLpep80/87/95/96/97
In this example, the assay was performed in both a two-day and three-day format. For the 2 day assay, 20,000 HEK293T cells were reverse-transfected in opaque 96-well assay plates with a total of 0.1 ng pF4A Ag FRB-NLpoly5P or FRB-NLpoly5A2 and pF4A Ag FKBP-NLpep80/87/95/96/97 using FuGENE HD at a DNA-to-FuGENE ratio of 1 to 8. pGEM-3Zf(+) DNA was added to bring total DNA in each transfection to 1 μg. 24 hours-post transfection, cells were washed with PBS and then incubated in phenol red-free OptiMEMI with or without 50 nM rapamycin for 2 h. 10 μM furimazine substrate (final concentration on cells) with or without 50 nM rapamycin in OptiMEMI was added directly to each well and incubated at room temperature for 5 min. Luminescence was then measured on a GloMax Multi with 0.5 s integration time.
For 3 day assay, 400,000 HEK293T cells were reverse-transfected with a total of 0.002 μg pF4A Ag FRB-NLpoly5P and pF4A Ag FKBP-NLpep80/87/95/96/97 using FuGENE HD at a DNA-to-FuGENE ratio of 1 to 8. pGEM-3Zf(+) DNA was added to bring total DNA in each transfection to 1 μg. 24-hours post-transfection, 10,000 cells were re-plated in opaque 96-well assay plates and incubated an additional 24 hours. Cells were washed with PBS and then incubated in phenol red-free OptiMEMI with or without 50 nM rapamycin for 2 h. 10 μM furimazine substrate (final concentration on cells) with or without 50 nM rapamycin in OptiMEMI was added directly to each well and incubated at room temperature for 5 min. Luminescence was then measured on a GloMax Multi with 0.5 s integration time. FIGS. 119 and 120 illustrate similar levels of luminescence in both the 2 day and 3 day assays. Assays performed with NLpoly5A2 showed greater rapamycin induction relative to NLpoly5P, and assays performed with NLpoly5A2 and NLpep96 showed greatest rapamycin induction of all tested combinations.
Example 73
Comparison of Luminescence Generated by Cells Expressing Different Combinations of FRB-NLpoly5A2 or FRB-NLpoly11S with FKBP-NLpep101/104/105/106/107/108/109/110
HEK293T cells (20,000) were reverse-transfected in opaque 96-well assay plates with a total of 0.1 ng pF4A Ag FRB-NLpoly5A2/11S and pF4A Ag FKBP-NLpep101/104/105/106/107/108/109/110 using FuGENE HD at a DNA-to-FuGENE ratio of 1 to 8. pGEM-3Zf(+) DNA was added to bring total DNA in each transfection to 1 μg. 24 hours-post transfection, cells were washed with PBS and then incubated in phenol red-free OptiMEMI with or without 50 nM rapamycin for 2 h. 10 μM furimazine substrate (final concentration on cells) with or without 50 nM rapamycin in OptiMEMI was added directly to each well and incubated at room temperature for 5 min. Luminescence was then measured on a GloMax Multi with 0.5 s integration time. FIG. 121 illustrates that, of tested combinations, NLpoly11S with NLpep101 showed the greatest rapamycin induction and one of the strongest rapamycin-specific luminescent signals.
Example 74
Comparison of Luminescence Generated by Cells Transfected with Different Combinations of FRB-NLpoly5A2 or FRB-NLpoly11S with FKBP-NLpep87/96/98/99/100/101/102/103
HEK293T cells (20,000) were reverse-transfected in opaque 96-well assay plates with a total of 0.1 ng pF4A Ag FRB-NLpoly5A2/11S and pF4A Ag FKBP-NLpep87/96/98/99/100/101/102/103 using FuGENE HD at a DNA-to-FuGENE ratio of 1 to 8. pGEM-3Zf(+) DNA was added to bring total DNA in each transfection to 1 μg. 24 hours-post transfection, cells were washed with PBS and then incubated in phenol red-free OptiMEMI with or without 50 nM rapamycin for 2 h. 10 μM furimazine substrate (final concentration on cells) with or without 50 nM rapamycin in OptiMEMI was added directly to each well and incubated at room temperature for 5 min. Luminescence was then measured on a GloMax Multi with 0.5 s integration time. FIG. 122 illustrates that the NLpoly11S and NLpep101 combination produces the highest induction while maintaining high levels of specific luminescence.
Example 75
Comparison of Luminescence Generated by Cells Transfected with Different Levels of FRB-NLpoly11S and FKBP-NLpep87/101/102/107 DNA
HEK293T cells (20,000) were reverse-transfected in opaque 96-well assay plates with a total of 0.01, 0.1, 1, or 10 ng pF4A Ag FRB-NLpoly11S and pF4A Ag FKBP-NLpep87/101/102/107 using FuGENE HD at a DNA-to-FuGENE ratio of 1 to 8. pGEM-3Zf(+) DNA was added to bring total DNA in each transfection to 1 μg. 24 hours-post transfection, cells were washed with PBS and then incubated in phenol red-free OptiMEMI with or without 50 nM rapamycin for 1.5 h. 10 μM furimazine substrate (final concentration on cells) with or without 50 nM rapamycin in OptiMEMI was added directly to each well and incubated at room temperature for 5 min. Luminescence was then measured on a GloMax Multi with 0.5 s integration time. FIG. 123 illustrates NLpoly11S with NLpep101 produces the overall lowest luminescence in untreated samples at all tested DNA levels, and the combination maintains relatively high levels of luminescence in rapamycin-treated samples.
Example 76
Comparison of Luminescence Generated by Cells Transfected with Different Levels of FRB-NLpoly5A2 and FKBP-NLpep87/101/102/107 DNA
HEK293T cells (20,000) were reverse-transfected in opaque 96-well assay plates with a total of 0.01, 0.1, 1, or 10 ng pF4A Ag FRB-NLpoly5A2 and pF4A Ag FKBP-NLpep87/101/102/107 using FuGENE HD at a DNA-to-FuGENE ratio of 1 to 8. pGEM-3Zf(+) DNA was added to bring total DNA in each transfection to 1 μg. 24 hours-post transfection, cells were washed with PBS and then incubated in phenol red-free OptiMEMI with or without 50 nM rapamycin for 1.5 h. 10 μM furimazine substrate (final concentration on cells) with or without 50 nM rapamycin in OptiMEMI was added directly to each well and incubated at room temperature for 5 min. Luminescence was then measured on a GloMax Multi with 0.5 s integration time. FIG. 124 illustrates that NLpoly5A2 generates higher luminescence in untreated samples than NLpoly11S shown in example 75.
Example 77
Rapamycin Dose Response Curve Showing Luminescence of Cells Expressing FRB-NLpolySP and FKBP-NLpep80/87 DNA
HEK293T cells (400,000) were reverse-transfected with a total of 0.001 μg pF4A Ag FRB-NLpolySP and 0.001 μg pF4A Ag FKBP-NLpep80/NLpep87 using FuGENE HD at a DNA-to-FuGENE ratio of 1 to 8. pGEM-3Zf(+) DNA was added to bring total DNA in each transfection to 1 μg. 24-hours post-transfection, 10,000 cells were re-plated in opaque 96-well assay plates and incubated an additional 24 hours. Cells were washed with PBS and then incubated in phenol red-free OptiMEMI with 0 to 500 nM rapamycin for 2 h. 10 μM furimazine substrate (final concentration on cells) with 0 to 500 nM rapamycin in OptiMEM was added directly to each well and incubated at room temperature for 5 min. Luminescence was then measured on a GloMax Multi with 0.5 s integration time. Kd was calculated with GraphPad Prism version 5.00 for Windows. FIG. 125 illustrates a rapamycin-specific increase in luminescence.
Example 78
Rapamycin Dose Response Curve Showing Luminescence of Cells Expressing FRB-NLpoly5A2 and FKBP-NLpep87/101 DNA
HEK293T cells (20,000) were reverse-transfected in opaque 96-well assay plates with a total of 0.1 ng pF4A Ag FRB-NLpoly5A2/11S and pF4A Ag FKBP-NLpep87/101 using FuGENE HD at a DNA-to-FuGENE ratio of 1 to 8. pGEM-3Zf(+) DNA was added to bring total DNA in each transfection to 1 μg. 24 hours-post transfection, cells were washed with PBS and then incubated in phenol red-free OptiMEMI with 0 to 1 μM rapamycin for 1.5 h. 10 μM furimazine substrate (final concentration on cells) with 0 to 1 μM rapamycin in OptiMEMI was added directly to each well and incubated at room temperature for 5 min. Luminescence was then measured on a GloMax Multi with 0.5 s integration time. FIG. 126 illustrates a sigmoidal dose response to rapamycin with NLpoly5A2/NLpep101 and NLpoly11S/NLpep101 combinations. While combinations with NLpep87 show an increase in luminescence with rapamycin, the collected data points deviate more from the sigmoidal curve.
Example 79
Comparison of Luminescence Generated by Cells Expressing FRB-11S and FKBP-101 and Treated with Substrate PBI-4377 or Furimazine
HEK293T cells (20,000) were reverse-transfected in opaque 96-well assay plates with a total of 0.1/1/10 ng pF4A Ag FRB-NLpoly11S and pF4A Ag FKBP-NLpep101 using FuGENE HD at a DNA-to-FuGENE ratio of 1 to 8. pGEM-3Zf(+) DNA was added to bring total DNA in each transfection to 1 μg. 24 hours-post transfection, cells were washed with PBS and then incubated in phenol red-free OptiMEMI with 0 or 50 nM rapamycin for 1.5 h. 10 μM furimazine or PBI-4377 substrate (final concentration on cells) with 0 to 50 nM rapamycin in OptiMEMI was added directly to each well and incubated at room temperature for 5 min. Luminescence was then measured on a GloMax Multi with 0.5 s integration time. FIG. 127 illustrates a decrease in luminescence and fold induction with the PBI-4377 substrate compared to the furimazine substrate.
Example 80
Time Course of Cells Expressing FRB-NLpoly11S/5A2 and FKBP-NLpep87/101 Conducted in the Presence or Absence of Rapamycin
HEK293T cells (20,000) were reverse-transfected in opaque 96-well assay plates with a total of 0.1 ng pF4A Ag FRB-NLpoly 11S/5A2 and pF4A Ag FKBP-NLpep87/101 using FuGENE HD at a DNA-to-FuGENE ratio of 1 to 8. pGEM-3Zf(+) DNA was added to bring total DNA in each transfection to 1 μg. 24 hours-post transfection, cells were washed with PBS and then phenol red-free OptiMEMI with 0 or 50 nM rapamycin and 10 μM furimazine was added either manually or via instrument injection. Luminescence was immediately measured on a GloMax Multi with 0.5 s integration time. FIGS. 128 and 129 illustrate that, of all combinations tested, NLpoly11S with NLpep101 has the lowest luminescence at time 0, hits a luminescent plateau faster and has the largest dynamic range.
Example 81
Luminescence Generated by FRB-NLpoly11S and FKBP-NLpep101 as Measured on Two Different Instruments
HEK293T cells (20,000) were reverse-transfected in opaque 96-well assay plates with a total of 0.1 ng pF4A Ag FRB-NLpoly11S and pF4A Ag FKBP-NLpep101 using FuGENE HD at a DNA-to-FuGENE ratio of 1 to 8. pGEM-3Zf(+) DNA was added to bring total DNA in each transfection to 1 μg. 24 hours-post transfection, cells were washed with PBS and then phenol red-free OptiMEMI with 0 or 50 nM rapamycin was added for 20 min. 10 μM furimazine (final concentration on cells) in OptiMEMI with 0 or 50 nM rapamycin was added and incubated for an additional 5 min. Luminescence was immediately measured on a GloMax Multi with 0.5 s integration time and on the Varioskan Flash with 450 nM band pass filter. FIG. 130 illustrates that the rapamycin-specific induction of FRB-NLpoly11S and FKBP-NLpep101 can be measured on different instruments.
Example 82
Images Showing Luminescence of Cells Expressing FRB-NLpoly11S and FKBP-NLpep101 at Various Times after Treatment with Rapamycin
HeLa cells (500,000) were reverse transfected with 1 μg pF4 Ag FRB-NLpoly11S and 1 μg pF4 Ag FKBP-NLpep101 using FuGENE HD at a DNA to FuGENE ratio of 1 to 4. Cells were transfected in 35 mm glass bottom culture dishes (MatTek #p35gc-1.5-14-C). 24 hours post-transfection, cells were washed with PBS and then incubate with 10 μM furimazine in OptiMEM for 5 min. 50 nM rapamycin in OptiMEMI was added to cells and luminescent images were acquired with LV200 at 10 s intervals for a total of 20 min. Instrument was at 37° C., objective was 60×, gain was 200 and exposure was 600 ms. FIG. 131 illustrates that imaging can detect an increase in cellular luminescence in cells expressing FRB-NLpoly11S and FKBP-NLpep101 following rapamycin treatment.
Example 83
Quantitation of the Signal Generated by Individual Cells Expressing FRB-NLpoly11S and FKBP-NLpep101 at Various Times after Treatment with Rapamycin
HeLa cells (500,000) were reverse transfected with 1 μg pF4 Ag FRB-NLpoly11S and 1 μg pF4 Ag FKBP-NLpep101 using FuGENE HD at a DNA to FuGENE ratio of 1 to 4. Cells were transfected in 35 mm glass bottom culture dishes (MatTek #p35gc-1.5-14-C). 24 hours post-transfection, cells were washed with PBS and then incubate with 10 μM furimazine in OptiMEM for 5 min. 50 nM rapamycin in OptiMEMI was added to cells, and luminescent images were acquired with LV200 at 10 s intervals for a total of 20 min. Instrument was at 37° C., objective was 60×, gain was 200, and exposure was 600 ms. The signal intensity of every cell in the field of view was analyzed with Image J software over the entire time period. FIG. 132 illustrates that signal generated by individual cells can be measured and that the increase in signal by each cell parallels the increase observed in the 96-well plate assay shown in FIGS. 128 and 129.
Example 84
Comparison of Luminescence in Different Cell Lines Expressing FRB-NLpoly11S and FKBP-NLpep101
HEK293T, HeLa, or U2-OS cells (20,000) were reverse-transfected in opaque 96-well assay plates with a total of 0.1 ng pF4A Ag FRB-NLpoly11S and pF4A Ag FKBP-NLpep101 using FuGENE HD at a DNA-to-FuGENE ratio of 1 to 8. pGEM-3Zf(+) DNA was added to bring total DNA in each transfection to 1 μg. 24 hours-post transfection, cells were washed with PBS and then phenol red-free OptiMEMI with 0 or 50 nM rapamycin was added for 20 min. 10 μM furimazine (final concentration on cells) in OptiMEMI with 0 or 50 nM rapamycin was added and incubated for an additional 5 min. Luminescence was immediately measured on a GloMax Multi with 0.5 s integration time. FIG. 133 illustrates similar levels of luminescence generated in the absence and presence of rapamycin in three different cells lines transfected with FRB-NLpoly11S and FKBP-NLpep101.
Example 85
Comparison of Luminescence Generated by Cells Expressing FRB-NLpoly11S and FKBP-NLpep101 after Treatment with the Rapamycin Competitive Inhibitor FK506
HEK293T cells (20,000) were reverse-transfected in opaque 96-well assay plates with a total of 0.1 ng pF4A Ag FRB-NLpoly11S and pF4A Ag FKBP-NLpep101 using FuGENE HD at a DNA-to-FuGENE ratio of 1 to 8. pGEM-3Zf(+) DNA was added to bring total DNA in each transfection to 1 μg. 24 hours-post transfection, cells were washed with PBS and then phenol red-free OptiMEMI with 0 or 20 nM rapamycin was added for 20 min. FK506 inhibitor in OptiMEM was added to cell at final concentration of 5 μM and incubated for 3 or 5 hours. Furimazine in OptiMEM was added to cells for a final concentration of 10 μM on cells. Luminescence was immediately measured on a GloMax Multi with 0.5 s integration time. FIG. 134 illustrates a decrease in rapamycin-induced luminescence after treatment with the competitive inhibitor FK506.
Example 86
Luminescence Generated by Cells Expressing FRB-NLpoly11S and FKBP-NLpep101 after Treatment with the Rapamycin Competitive Inhibitor FK506
HEK293T cells (20,000) were reverse-transfected in opaque 96-well assay plates with a total of 0.1 ng pF4A Ag FRB-NLpoly11S and pF4A Ag FKBP-NLpep101 using FuGENE HD at a DNA-to-FuGENE ratio of 1 to 8. pGEM-3Zf(+) DNA was added to bring total DNA in each transfection to 1 μg. 24 hours-post transfection, cells were washed with PBS and then phenol red-free OptiMEMI with 0 or 20 nM rapamycin was added for 2.5 hours. FK506 inhibitor in OptiMEM was added to cell via injector at final concentration of 0, 1 or 10 μM in OptiMEM with 10 μM. Luminescence was measured every 10 min for 4 hours on a GloMax Multi set to 37° C. with 0.5 s integration time. FIG. 135 illustrates that by 200 s, FK506 inhibitor can reduce luminescence close to levels of untreated cells.
Example 87
Luminescence Generated by Cells Transfected with Different Combinations of V2R-NLpoly5A2 or V2R-NLpoly11S with NLpep87/101-ARRB2 in the Presence or Absence of the V2R Agonist AVP
HEK293T cells (20,000) were reverse-transfected in opaque 96-well assay plates with a total of 0.1, 1, or 10 ng pF4A Ag V2R-NLpoly11S and pF4A Ag ARRB2-NLpep87/101 using FuGENE HD at a DNA-to-FuGENE ratio of 1 to 8. pGEM-3Zf(+) DNA was added to bring total DNA in each transfection to 1 μg. 24 hours-post transfection, cells were washed with PBS and then phenol red-free OptiMEMI with 0 or 1 μM AVP and 10 μM furimazine was added for 25 min. Luminescence was then measured on a GloMax Multi with 0.5 s integration time.
FIG. 136 illustrates that V2R-NLpoly11S with NLpep101 generates the greatest AVP-specific increase in luminescence. Combinations with NLpep87 show no significant response to AVP.
Example 88
Time Course Showing Luminescence Generated by Cells Transfected with V2R-NLpoly5A2 or V2R-NLpoly11S and NLpep87/101-ARRB2 after Treatment with AVP
HEK293T cells (20,000) were reverse-transfected in opaque 96-well assay plates with a total of 0.1 or ing pF4A Ag V2R-NLpoly11S or ing pF4A Ag V2R-NLpoly5A2 and pF4A Ag ARRB2-NLpep87/101 using FuGENE HD at a DNA-to-FuGENE ratio of 1 to 8. pGEM-3Zf(+) DNA was added to bring total DNA in each transfection to 1 μg. 24 hours-post transfection, cells were washed with PBS and then phenol red-free OptiMEMI with 0 or 1 μM AVP and 10 μM furimazine was added either manually (FIG. 137) or via instrument injection (FIG. 138). Luminescence was then measured on a GloMax Multi every 5 min for 25 min with 0.5 s integration time at room temperature (FIGS. 137 and 138) or 37° C. (FIG. 139). FIGS. 137 and 138 illustrate a time-dependent increase in AVP-induced luminescence for V2R-NLpoly11S with NLpep101-ARRB2 that begins to peak at 600 s. Combinations with V2R-NLpoly5A2 and NLpep87 do not show a significant increase in luminescence over time. FIG. 139 illustrates that at 37° C. all NLpoly11S and NLpep101 combinations tested show a time-dependent increase in AVP-induced luminescence that levels out around 200 s.
Example 89
Comparison of Luminescence in Different Cell Lines Expressing V2R-NLpoly11S and NLpep101-ARRB2
HEK293T, HeLa, or U2-OS cells (20,000) were reverse-transfected in opaque 96-well assay plates with a total of ing pF4A Ag V2R-NLpoly11S and pF4A Ag ARRB2-NLpep87/101 using FuGENE HD at a DNA-to-FuGENE ratio of 1 to 8. pGEM-3Zf(+) DNA was added to bring total DNA in each transfection to 1 μg. 24 hours-post transfection, cells were washed with PBS and then phenol red-free OptiMEMI with 0 or 1 μM AVP was added for 20 min. Furimazine in OptiMEM was then added to a final concentration of 10 μM on cells, and luminescence was measured on a GloMax Multi with 0.5 s integration time.
FIG. 140 illustrates similar luminescence levels in three different cell lines expressing V2R-NLpoly11S and NLpep101-ARRB2 in the presence and absence of AVP.
Example 90
Luminescence of Cells Expressing V2R-NLpoly11S and NLpep101-ARRB2 at Various Times after Treatment with AVP
HeLa cells (500,000) were reverse transfected with 1 μg pF4 Ag V2R-NLpoly11S and 1 μg pF4 Ag ARRB2-NLpep101 using FuGENE HD at a DNA to FuGENE ratio of 1 to 4. Cells were transfected in 35 mm glass bottom culture dishes (MatTek #p35gc-1.5-14-C). 24 hours post-transfection, cells were washed with PBS and then incubate with 10 μM furimazine in OptiMEM for 5 min. 1 uM AVP in OptiMEMI was added to cells, and luminescent images were acquired with LV200 at 15 s intervals for a total of 30 min. Instrument was at 37° C., objective was 60× or 150×, gain was 600, and exposure was is or 2 s. FIGS. 141 and 142 illustrate that imaging can detect the increase in luminescence and formation of punctate in individual cells after treatment with AVP.
Example 91
Dissociation Constants for NLpeps
NLpoly 5P E. coli clarified lysate (prepared as described previously) was diluted 1:1,000 into PBS+0.1% Prionex. 4× concentrations of NLpep78-HT (E. coli clarified lysate prepared as described previously) were made in PBS+0.1% Prionex. 20 uL NLpoly 5P and 20 uL NLpep78 were mixed and shaken for 10 min at RT. 40 uL NanoGlo/Fz was added and shaken for 10 min at RT. Luminescence was measured on GloMax luminometer with 0.5 s integration. Kd was determined using Graphpad Prism, One site-specific binding, best-fit values. FIG. 80 compares the dissociation constants for an NLpep consisting of either 1 or 2 repeat units of NLpep78.
Example 92
Affinity Between NLpoly 5A2 and NLpep86
NLpoly 5A2 lysate (prepared as described previously after transfecting CHO cells) was diluted 1:10 into PBS+0.1% Prionex. 4× concentrations of NLpep86 (synthetic NLpep) were made in PBS+0.1% Prionex. 20 uL NLpoly and 20 uL NLpep were mixed and shaken for 10 min at RT. 40 uL NanoGlo/Fz was added and shaken for 10 min at RT. Luminescence was measured on GloMax luminometer with 0.5 s integration. Kd was determined using Graphpad Prism, One site-specific binding, best-fit values. FIG. 81 illustrates the affinity between NLpoly 5A2 and NLpep86.
Example 93
Luminescence of NLpoly Variants
A single colony of various NLpolys were inoculated individually into 200 uL minimal media and grown for 20 hrs at 37° C. on shaker. 10 uL of overnight culture was diluted into 190 uL fresh minimal media and grown for 20 hrs at 37° C. on shaker. 10 uL of this overnight culture was diluted into 190 uL auto-induction media (previously described) and grow for 18 hrs at 25° C. on shaker. 10 uL of the expression culture was mixed with 40 uL of assay lysis buffer (previously described) without NLpep or with NLpep78-HT (1:3,860 dilution) or NLpep79-HT (1:10,000 dilution). The mixtures were shaken for 10 min at RT, 50 uL NanoGlo+Fz added and shaken again for 10 min at RT. Luminescence was measured on a GloMax luminometer with 0.5 sec integration. FIG. 82 demonstrates the luminescence from NLpoly variants without an NLpep or with NLpep78 or NLpep79. The results show that the NLpoly variant 11S (12S-51) has improved luminescence over the other variants.
Example 94
Dissociation Constants and Vmax Values for NLpolys with 96 Variants of NLpeps
NLpeps were synthesized in array format by New England Peptide (peptides blocked at N-terminus by acetylation and at C-terminus by amidation; peptides in arrays were synthesized at  ̃1 mg scale) (Table 6). Each peptide was lyophilized in 3 separate plates. Each well from 1 of the 3 plates of peptides was dissolved in 100 uL nanopure water, and the A260 measured and used to calculate the concentration using the extinction coefficient of each peptide. The concentration was then adjusted based on the purity of the peptide, and nanopure water was added to give a final concentration of 750 uM.
Peptides were diluted to 12.66 uM (4×) in PBS+0.1% Prionex and then diluted serially 7 times (8 concentrations total) in 0.5 log steps (3.162 fold dilution). NLpolys 5P, 8S, 5A2 or 11S were diluted into PBS+0.1% Prionex as follows: 5P 1:2,000; 8S 1:10,000; 11S 1:150,000, 5A2 1:1,000. 25 uL each NLpep+25 uL each NLpoly were mixed and incubated for 30 min at RT. 50 uL NanoGlo+100 uM Fz was added and incubated for 30 min at RT. Luminescence was measure on a GloMax Multi+ with 0.5 sec integration. Kd/Vmax were determined using Graphpad Prism, One site-specific binding, best-fit values. FIGS. 83-90 illustrate the dissociation constant and Vmax values from NLpolys with the 96 variant NLpeps. The results indicate specific mutations in the NLpeps that exhibit lower binding affinity without loss in Vmax.







TABLE 6







Peptide Array 1












Sequence
SEQ ID NO.







array1.1
VTGWRLCERIL
2366



 



array1.2
VSGWRLFKKIS
2367



 



array1.3
VTGYRLFKKIS
2368



 



array1.4
ISGWRLFKKIS
2369



 



array1.5
ASGWRLFKKIS
2370



 



array1.6
GSGWRLFKKIS
2371



 



array1.7
KSGWRLFKKIS
2372



 



array1.8
LSGWRLFKKIS
2373



 



array1.9
QSGWRLFKKIS
2374



 



array1.10
SSGWRLFKKIS
2375



 



array1.11
TSGWRLFKKIS
2376



 



array1.12
VVGWRLFKKIS
2377



 



array1.13
VKGWRLFKKIS
2378



 



array1.14
VIGWRLFKKIS
2379



 



array1.15
VEGWRLFKKIS
2380



 



array1.16
VAGWRLFKKIS
2381



 



array1.17
VQGWRLFKKIS
2382



 



array1.18
VHGWRLFKKIS
2383



 



array1.19
VSAWRLFKKIS
2384



 



array1.20
VSSWRLFKKIS
2385



 



array1.21
VSGFRLFKKIS
2386



 



array1.22
VSGWKLFKKIS
2387



 



array1.23
VSGWQLFKKIS
2388



 



array1.24
VSGWELFKKIS
2389



 



array1.25
VSGWALFKKIS
2390



 



array1.26
VSGWRIFKKIS
2391



 



array1.27
VSGWRVFKKIS
2392



 



array1.28
VSGWRTFKKIS
2393



 



array1.29
VSGWRYFKKIS
2394



 



array1.30
VSGWRKFKKIS
2395



 



array1.31
VSGWRFFKKIS
2396



 



array1.32
VSGWRLAKKIS
2397



 



array1.33
VSGWRLDKKIS
2398



 



array1.34
VSGWRLEKKIS
2399



 



array1.35
VSGWRLGKKIS
2400



 



array1.36
VSGWRLHKKIS
2401



 



array1.37
VSGWRLIKKIS
2402



 



array1.38
VSGWRLKKKIS
2403



 



array1.39
VSGWRLLKKIS
2404



 



array1.40
VSGWRLMKKIS
2405



 



array1.41
VSGWRLNKKIS
2406



 



array1.42
VSGWRLQKKIS
2407



 



array1.43
VSGWRLRKKIS
2408



 



array1.44
VSGWRLSKKIS
2409



 



array1.45
VSGWRLTKKIS
2410



 



array1.46
VSGWRLVKKIS
2411



 



array1.47
VSGWRLWKKIS
2412



 



array1.48
VSGWRLYKKIS
2413



 



array1.49
VSGWRLFEKIS
2414



 



array1.50
VSGWRLFVKIS
2415



 



array1.51
VSGWRLFSKIS
2416



 



array1.52
VSGWRLFRKIS
2417



 



array1.53
VSGWRLFTKIS
2418



 



array1.54
VSGWRLFNKIS
2419



 



array1.55
VSGWRLFQKIS
2420



 



array1.56
VSGWRLFKRIS
2421



 



array1.57
VSGWRLFKQIS
2422



 



array1.58
VSGWRLFKEIS
2423



 



array1.59
VSGWRLFKAIS
2424



 



array1.60
VSGWRLFKKVS
2425



 



array1.61
VSGWRLFKKLS
2426



 



array1.62
VSGWRLFKKAS
2427



 



array1.63
VSGWRLFKKFS
2428



 



array1.64
VSGWRLFKKES
2429



 



array1.65
VSGWRLFKKTS
2430



 



array1.66
VSGWRLFKKIL
2431



 



array1.67
VSGWRLFKKIA
2432



 



array1.68
VSGWRLFKKIE
2433



 



array1.69
VSGWRLFKKIV
2434



 



array1.70
VSGWRLFKKIG
2435



 



array1.71
VSGWRLFKKIH
2436



 



array1.72
VSGWRLFKKIT
2437



 



array1.73
VVGYRLFKKIS
2438



 



array1.74
VKGYRLFKKIS
2439



 



array1.75
VIGYRLFKKIS
2440



 



array1.76
VEGYRLFKKIS
2441



 



array1.77
VAGYRLFKKIS
2442



 



array1.78
VQGYRLFKKIS
2443



 



array1.79
VHGYRLFKKIS
2444



 



array1.80
VTAYRLFKKIS
2445



 



array1.81
VTSYRLFKKIS
2446



 



array1.82
VTGYRIFKKIS
2447



 



array1.83
VTGYRVFKKIS
2448



 



array1.84
VTGYRTFKKIS
2449



 



array1.85
VTGYRYFKKIS
2450



 



array1.86
VTGYRKFKKIS
2451



 



array1.87
VTGYRFFKKIS
2452



 



array1.88
ISGWRLMKNIS
2453



 



array1.89
ASGWRLMKKES
2454



 



array1.90
VSGWRLMKKVS
2455



 



array1.91
ISGWRLLKNIS
2456



 



array1.92
ASGWRLLKKES
2457



 



array1.93
VSGWRLLKKVS
2458



 



array1.94
ISGWRLAKNIS
2459



 



array1.95
ASGWRLAKKES
2460



 



array1.96
VSGWRLAKKVS
2461










Example 95
Solubility of NLpoly Variants
A single NLpoly 5A2, 12S, 11S, 12S-75, 12S-107 or 5P-B9 colony was inoculated into 5 mL LB culture and incubated at 37° C. overnight with shaking. The overnight culture was diluted 1:100 into fresh LB and incubated at 37° C. for 3 hrs with shaking. Rhamnose was added to the cultures to 0.2% and incubated 25° C. overnight with shaking. 900 ul of these overnight cultures were mixed with 100 uL 10× FastBreak Lysis Buffer (Promega Corporation) and incubated for 15 min at RT. A 75 uL aliquot (total) was removed from each culture and saved for analysis. The remaining culture from each sample was centrifuged at 14,000×rpm in a benchtop microcentrifuge at 4° C. for 15 min. A 75 uL aliquot of supernatant (soluble) was removed from each sample and saved for analysis. 25 uL of 4×SDS buffer was added to the saved aliquots and incubated at 95° C. for 5 min. 5 ul of each sample was loaded onto a 4-20% Tris-Glycine SDS gel and run at  ̃190V for  ̃50 min. The gel was stained with SimplyBlue Safe Stain and imaged on a LAS4000. FIG. 91 shows a protein gel of total lysates and the soluble fraction of the same lysate for the NLpoly variants. With the exception of 5A2, all variants exhibit a percentage of NLpoly in the soluble fraction.
Example 96
Solubility and Dissociation Constant of NLpoly Variants
A single NLpoly colony (listed in FIG. 92) was inoculated into 5 mL LB culture and incubated at 37° C. overnight with shaking. The overnight culture was diluted 1:100 into fresh LB and incubated at 37° C. for 3 hrs with shaking. Rhamnose was added to the cultures to 0.2% and incubated 25° C. overnight with shaking. 900 ul of these overnight cultures were mixed with 100 uL 10× FastBreak Lysis Buffer (Promega Corporation) and incubated for 15 min at RT. A 75 uL aliquot (total) was removed from each culture and saved for analysis. The remaining culture from each sample was centrifuged at 14,000×rpm in a benchtop microcentrifuge at 4° C. for 15 min. A 75 uL aliquot of supernatant (soluble) was removed from each sample and saved for analysis. 25 uL of 4×SDS buffer was added to the saved aliquots and incubated at 95° C. for 5 min. 5 ul of each sample was loaded onto a 4-20% Tris-Glycine SDS gel and run at  ̃190V for  ̃50 min. The gel was stained with SimplyBlue Safe Stain and imaged on a LAS4000.
FIG. 92 shows a protein gel of total lysates and the soluble fraction of the same lysate for NLpoly variants as well a table containing the dissociation constants for the same variants.
Example 97
Substrate Specificity for NLpoly 5P and 11S with NLpep79
E. coli clarified lysates were prepared for NLpoly 5P or 11S as described previously. The NLpoly lysates were then serially diluted in steps of 10-fold into PBS+0.1% Prionex. 25 uL NLpoly and 25 uL synthetic NLpep79 (400 nM, 4×) were mixed and incubated for 10 min at RT. 50 uL NanoGlo+100 uM Fz was added, incubated for 10 min at RT, luminescence measured on a GloMax Multi+ with 0.5 sec integration. FIG. 93 shows the substrate specificity for 5P and 11S with NLpep79 and demonstrates that 11S has superior specificity for furimazine than 5P.
Example 98
Solubility of NLpoly Variants from Various Steps of Evolution
A single NLpoly WT, 5A2, 5P, 8S or 11S colony was inoculated into 5 mL LB culture and incubated at 37° C. overnight with shaking. The overnight culture was diluted 1:100 into fresh LB and incubated at 37° C. for 3 hrs with shaking. Rhamnose was added to the cultures to 0.2% and incubated 25° C. overnight with shaking. 900 ul of these overnight cultures were mixed with 100 uL 10× FastBreak Lysis Buffer (Promega Corporation) and incubated for 15 min at RT. A 75 uL aliquot (total) was removed from each culture and saved for analysis. The remaining culture from each sample was centrifuged at 14,000×rpm in a benchtop microcentrifuge at 4° C. for 15 min. A 75 uL aliquot of supernatant (soluble) was removed from each sample and saved for analysis. 25 uL of 4×SDS buffer was added to the saved aliquots and incubated at 95° C. for 5 min. 5 ul of each sample was loaded onto a 4-20% Tris-Glycine SDS gel and run at  ̃190V for  ̃50 min. The gel was stained with SimplyBlue Safe Stain and imaged on a LAS4000.
FIG. 104 shows a protein gel of total lysates and the soluble fraction of the same lysate for NLpoly variants from various steps of the evolution process. These results demonstrate that the solubility of NLpoly was dramatically increased in the evolution process.
Example 99
Chemical Labeling of Proteins
The non-luminescent peptides (NLpeps) of the present invention can be used to chemically label proteins. An NLpep of the present invention can be synthesized to contain a reactive group, e.g., biotin, succinimidyl ester, maleimide, etc., and attached (e.g., conjugated, linked, labeled, etc.) to a protein, e.g., antibody. The NLpep-labeled protein, e.g., NLpep-antibody, can then be used in a variety of applications, e.g., ELISA. The interaction/binding of the NLpep-labeled protein, e.g., NLpep-antibody, to its target/binding partner would be detected by adding an NLpoly of the present invention and NanoGlo® assay reagent. The luminescence generated by the interaction of the NLpep-labeled protein and NLpoly would correlate to the interaction of the NL-labeled protein to its target/binding partner. This concept could allow for multiple NLpeps to be attached to a single protein molecule thereby resulting in multiple NLpep-labeled protein/NLpoly interactions leading to signal amplification.
Example 100
Detection of Post-Translational Protein Modification Using HaloTag-NLpep by Western Blotting
Several proteins can be posttranslationally modified by AMPylation or ADP-ribosylation. In AMPylation, AMP is added to the target protein by a phosphodiester bond using ATP as the donor molecule. Similarly, in ADP-ribosylation, an ADP-ribose moiety is added to target proteins through a phosphodiester bond using NAD+ as the donor molecule. It has been shown that the N6-position of both ATP and NAD+ can be used to tag linkers without affecting the posttranslational event. If a N6-modified chloroalkane-ATP or -NAD+ is used to perform the AMPylation or ADP-ribosylation reaction, the target proteins would be modified to contain the chloroalkane-ATP or -NAD+.
The N6-modified ATP/NAD has been used in combination with click-chemistry to develop in-gel fluorescent-based detection systems. Detection of these post-translational modifications by western blotting techniques requires antibodies, which are often not specific or not available. An alternative approach could be to combine the properties of HaloTag® technology and the high luminescence of NanoLuc® luciferase (NL). Upon post-translational modification of target proteins with chloroalkane-ATP (for AMPylation) or chloroalkane-NAD+ (for ADP-ribosylation) using either cell lysate or purified proteins, samples can be resolved by SDS-PAGE and transferred to PVDF membrane. Following blocking, the blot can be incubated with HaloTag-NLpep. HaloTag will bind to the post-translationally-modified proteins. In the next step, the NLpoly and furimazine could be added to the blot to detect the bioluminescence. This detection method is an alternative to a chemiluminescent-based approach for detection of western blots. A chemiluminescent-based approach could involve incubation HaloTag-protein G fusions (as a primary) in the next step any secondary antibody-linked to HRP could be used followed by ECL reaction.
Example 101
Post Translational Modification Assays
Post translational modifications (PTMs) of proteins are central to all aspects of biological regulation. PTMs amplify the diverse functions of the proteome by covalently adding functional groups to proteins. These modifications include phosphorylation, methylation, acetylation, glycosylation, ubiquitination, nitrosylation, lipidation and influence many aspects of normal cell biology and pathogenesis. More specifically, histone related PTMs are of great importance. Epigenetic covalent modifications of histone proteins have a strong effect on gene transcriptional regulation and cellular activity. Examples of post translational modification enzymes include but not limited to, Kinases/Phosphatases, Methyltransferases (HMT)/Demethylases (HDMT), Acetyltransferases/Histone Deacetylases, Glycosyltransferases/Glucanases and ADP-Ribosyl Transferases. Under normal physiological conditions, the regulation of PTM enzymes is tightly regulated. However, under pathological conditions, these enzymes activity can be dysregulated, and the disruption of the intracellular networks governed by these enzymes leads to many diseases including cancer and inflammation.
The non-luminescent peptides (NLpep) and non-luminescent polypeptides (NLpoly) of the present invention can be used to determine the activity of PTM enzymes by monitoring changes in covalent group transfer (e.g. phosphoryl, acetyl) to a specific peptide substrate linked to an NLpep of the present invention. The NLpep will be linked through peptide synthesis to small PTM enzyme specific peptide and used as a substrate for the PTM enzyme.
A) PTM Transferase Assays (HAT)
Once the PTM enzyme reaction has occurred, an aminopeptidase can be used to degrade the non-modified peptide (NLpep; control). The modified (acetylated) peptide (NLpep-PTM enzyme substrate) would be degraded at a very slow rate or would not be degraded at all as the aminopeptidase activity is known to be affected by a PTM. Once the aminopeptidase reaction is complete, the NLpoly is added with the NanoGlo® assay reagent containing Furimazine. Luminescence would be generated from the sample where PTM occurred via the interaction of the NLpep and NLpoly. If no PTM occurred, the NLpep would be degraded, and no interaction between the NLpep and NLpoly would occur, thereby no luminescence would be generated. This concept is exemplified in FIG. 197 for a general transferase enzyme concept and in FIG. 145 for H3K4/9 acetyltransferases.
The reaction would be performed under optimal enzyme reaction condition using the histone peptide substrate linked to NLpep of the present invention and Acetyl-CoA or SAM as the acetyl or methyl group donor. A buffer containing aminopeptidase or a mixture of aminopeptidases would be added to degrade specifically all the non-modified substrates. A buffer containing a NLpoly of the present invention and an aminopeptidase inhibitor would be added. NanoGlo® assay reagent would be added, and luminescence detected. Luminescence generated would be proportional to the amount of non-degraded NLpep present, and therefore would correlate with the amount of methylated or acetylated substrates, thereby indicating the amount of methyl or acetyl transferase activity. The assay can also be applied to PTM such as phosphorylation, glycosylation, ubiquitination, nitrosylation, and lipidation.
B) PTM Hydrolase Assays (HDMT)
In a similar concept to A) can be used for Histone Demethylases (HDMT). However, instead of an aminopeptidase, a PTM-specific antibody can be used to create activity interference. An NLpep of the present invention could be linked through peptide synthesis to small methylated peptide and used as a substrate for the hydrolase. Once a hydrolase reaction has been completed, an anti-methyl antibody can be added to the reaction. This antibody will bind specifically to the methylated peptide (control). The peptide product generated by the HDMT will not bind to the antibody. Then, an NLpoly of the present invention can be added. If the antibody interferes with the interaction of NLpep and NLpoly, no luminescence will be generated. If there was hydrolysis of the PTM by the demethylase, the NLpep and NLpoly will interact, and luminescence will be generated. This concept is exemplified in FIG. 198 for a general hydrolase enzyme concept and in FIG. 146 H3K4/9 demethylases.
The concept of aminopeptidase degradation of the non-modified substrate can also be used for a hydrolase assay except it would be a loss of signal assay instead of a gain of signal. The reaction would be performed under optimal enzyme reaction condition using a modified (methylated or acetylated) histone peptide substrate linked to an NLpep of the present invention. A buffer containing an antibody capable of recognizing the methyl or acetyl group would be added. A buffer containing an NLpoly of the present invention would be added. The NLpoly would interact with NLpep not bound to the antibody. NanoGlo® assay reagent would be added, and luminescence detected. The luminescence generated would be proportional to the amount of NLpep not bound to the antibody, and therefore would correlate with the amount of demethylated or deacetylated substrate, thereby indicating the amount of demethylase or deacetylase activity. Both hydrolase assay concepts can also be applied to PTM hydrolases such as phosphatases, glucanases and deubiquitinases.
In another version of these concepts, the PTM transfer or hydrolysis on the peptide-NLpep would be alone sufficient to reduce or enhance the interaction of NLpep with NLpoly and therefore decrease or increase the luminescence signal without the need of aminopeptidase or antibody.
The method of the present invention was used to assay a representative transferase, the Tyrosine Kinase SRC using the following NLpep-SRC substrate peptide: YIYGAFKRRGGVTGWRLCERILA (SEQ ID NO: 2586). SRC enzyme was titrated in 10 μl Reaction Buffer A (40 mM Tris 7.5, 20 mM MgCl2 and 0.1 mg/ml BSA) in the presence of 150 μM ATP and 2.5 μM NLpep-Src substrate and incubated for 1 hour at 23° C. After incubation, 10 μl of Amino-peptidase M (APM) reagent (40 mM Tris 7.5, 0.1 mg/ml BSA and 50 mU APM) was added, mixed for 2 minutes on an orbital shaker, and then incubated at 37° C. for 2 hours. To the samples, 30 μl of NLpoly Reagent was added, and the samples were incubated at room temperature. NLpoly Reagent contained the NLpoly fragment and an Aminopeptidase inhibitor. After 30 minutes, 50 μl NanoGlo® assay reagent was added and the luminescence was recorded after 3 minutes on a luminometer. It was found that an increase in SRC kinase enzyme activity is correlated with an increase in luminescence over background (FIG. 199). Only background activity was found when SRC was not present indicating that the non-phosphorylated NLpep-SRC substrate peptide was digested resulting in no light production by the NLpoly fragment, thus demonstrating use of the method of the present invention to monitor the activity of a transferase enzyme such as a kinase.
Example 102
Detection of Specific RNAs (Noncoding RNA or mRNA) of Interest in Mammalian Cells, Cell Lysate or Clinical Sample
The non-luminescent peptide (NLpep) and non-luminescent polypeptide (NLpoly) of the present invention can be tethered to an RNA binding domain (RBD) with engineered sequence specificity. The specificity of the RBD can be changed with precision by changing unique amino acids that confers the base-specificity of the RBD. An example of one such RBD is the human pumilio domain (referred here as PUM). The RNA recognition code of PUM has been very well established. PUM is composed of eight tandem repeats (each repeat consists of 34 amino acids which folds into tightly packed domains composed of alpha helices). Conserved amino acids from the center of each repeat make specific contacts with individual bases within the RNA recognition sequence (composed of eight bases). The sequence specificity of the PUM can be altered precisely by changing the conserved amino acid (by site-directed mutagenesis) involved in base recognition within the RNA recognition sequence. For detection of specific RNAs in the cell, PUM domains (PUM1 and PUM2) with customized sequence specificities for the target RNA can be tethered to a NLpep and NLpoly of the present invention (e.g., as a genetic fusion protein via genetic engineering) and can be expressed in mammalian cells. PUM1 and PUM2 are designed to recognize 8-nucleotide sequences in the target RNA which are proximal to each other (separated by only few base pairs, determined experimentally). Optimal interaction of PUM1 and PUM2 to their target sequence is warranted by introducing a flexible linker (sequence and length of the linker to be determined experimentally) that separates the PUM and the non-luminescent peptide and non-luminescent polypeptide. Binding of the PUM1 and PUM2 to their target sequence will bring the NLpep and NLpoly into close proximity in an orientation that results in a functional complex formation capable of generating bioluminescent signal under our specific assay condition. A functional bioluminescent complex would not be generated in the absence of the RNA target due to the unstable interaction of the NLpep and NLpoly pairs that constitutes the complex.
A similar strategy can also be used for detecting RNA in clinical sample in vitro. The NLpep-PUM fusion proteins with customized RNA specificity can be expressed and purified from suitable protein expression system (such as E. coli or mammalian protein expression system). Purified components can be added to the biological sample along with suitable substrate and assay components to generate the bioluminescent signal.
Example 103
DNA Oligo-Based Detection of Specific RNA (Noncoding RNA or mRNA) in Clinical Sample or Mammalian Cell Lysate
A non-luminescent peptide (NLpep) and non-luminescent polypeptide (NLpoly) of the present invention can be attached to oligonucleotides complementary to the target RNA with suitable linker (amino acids or nucleotides). Functional assembly of bioluminescent complex occurs only when sequence specific hybridization of DNA oligo to their target RNA brings the NLpep and NLpoly into close proximity in an ideal conformation optimal for the generation of a bioluminescent signal under the assay conditions. The detection can also be achieved through a three-component complementation system involving two NLpeps and a third NLpoly. For example, two NLpep-DNA conjugates will be mixed with the target RNA. Functional assembly of the bioluminescent complex is achieved by subsequent addition of the third NLpoly. Thus, if a detectable signal is produced under specific assay conditions using a clinical sample or cell lysate, the presence of target RNA in such a sample is inferred. Such assays are useful for detecting RNAs derived from infectious agents (viral RNAs) and specific RNA biomarkers (implicated in many disease conditions such as various forms of cancers, liver diseases, and heart diseases), and could provide a new avenue for diagnosis and prognosis of many disease conditions.
Example 104
In-Vivo Imaging
Biotechnology-derived products (Biologics), including antibodies, peptides and proteins, hold great promises as therapeutics agents. Unlike small molecule drugs, biologics are large molecules with secondary and tertiary structures and often contain posttranslational modifications. Internalization, intracellular trafficking, bio-distribution, pharmacokinetics and pharmacodynamics (PK/PD), immunogenicity, etc. of biologics differ significantly from small molecule drugs, and there is a need for new tools to ‘track’ these antibodies in vivo. Conventional chemical labeling with enzyme reporters (HRP, luciferase, etc.) or small fluorescent tags can significantly alter the therapeutic value of the biologics and are not ideal for in vivo imaging using biologics. Radioisotope-labeling for PET-based imaging is also not convenient.
The NLpolys and NLpeps described herein offer a novel solution for in vivo imaging of biologics. The NLpep can be genetically encoded into a biologic therapeutics without any synthetic steps. Genetic encoding allows precise control over amount of peptide per biologic molecule as well as its position, thereby minimizing any perturbation to its therapeutic value. For imaging, a NLpoly along with substrate, e.g., furimazine, can be injected into the animal. If the NLpep-biologic and NLpoly interact, luminescence would be generated. Alternatively, a transgenic animal expressing NLpoly can be used as a model system.
Example 105
BRET Applications
This concept fundamentally measures three moieties coming together. Two of the NLpolys and/or NLpeps form a complex, and the third moiety, which is either fluorescent or bioluminescent, provides an energy transfer component. If the complex formed is bioluminescent, both bioluminescence and energy transfer (i.e., BRET) can be measured. If the complex formed is fluorescent, the magnitude of energy transfer can be measured if the third component is a bioluminescent molecule.
A) This example demonstrates a fluorescent dye attached to a NLpep. Alternatively, a fluorescent protein could be fused, e.g., a fusion protein, with a NLpoly or NLpep (created from a genetic construct).
E. coli clarified lysate of NLpoly WT was prepared as described previously. 40 uL NLpoly WT lysate was mixed with 10 uL of PBI-4730 (NLpep1) or PBI-4877 (NLpep1-TMR) and incubated for 10 min at RT. 50 uL 100 uM furimazine in 50 mM HEPES pH 7.4 was added and incubated for 30 min at RT. Luminescence was measured over 400-700 nm on TECAN M1000.
FIG. 147 illustrates very efficient energy transfer from the NLPoly/NLPep complex (donor) to TMR (acceptor), and the corresponding red shift in the wavelength of light being emitted.
B) This example demonstrates using the BRET in detection, such as detecting small molecule concentration or enzymatic activity. Because energy transfer is strongly dependent on distance, the magnitude of energy transfer can often be related to the conformation of the system. For instance, insertion of a polypeptide that chelates calcium can be used to measure calcium concentration through modulation of energy transfer.
An enzyme that also changes the distance, either through causing a conformational change of the sensor as above or through cleavage of the sensor from the fluorescent moiety, can be measured through a system as described herein. A NLpoly or NLpep bound to a fluorescent moiety gives energy transfer when the NLpoly and NLpep interact. One example of this is a peptide sensor that has been made wherein the NLpep is conjugated to a fluorescent TOM dye via a DEVD linker (Caspase-3 cleavage site). When exposed to the NLpoly, energy transfer is observed. When exposed to Caspase-3, energy transfer is eliminated, but luminescence at 460 nm remains.
NLpoly 5A2 and NL-HT (NanoLuc fused to HaloTag) were purified. 20 uL of 8 pM NL-HT was mixed with 20 uL of 100 nM PBI-3781 (See, e.g., U.S. patent application Ser. No. 13/682,589, herein incorporated by reference in its entirety) and incubated for 10 min at RT. 40 uL NanoGlo+100 uM furimazine was added, and luminescence measured over 300-800 nm on TECAN M1000.
20 uL of 33 ng/uL NLpoly 5A2 was mixed with 20 uL of −500 uM PBI-5074 (TOM-NCT-NLpep). 40 uL NanoGlo+100 uM furimazine was added, and luminescence measured over 300-800 nm on TECAN M1000.
FIG. 148 illustrates energy transfer from the NLPoly/NLPep complex (donor) to TOM-dye (acceptor), and the corresponding red shift in the wavelength of light being emitted.
C) Ternary Interactions
The energy transfer with an NLpoly and NLpep can also be used to measure three molecules interacting. One example would be a GPCR labeled with NLpoly and a GPCR interacting protein with NLpep that forms a bioluminescent complex when they interact. This allows measurement of the binary interaction. If a small molecule GPCR ligand bearing an appropriate fluorescent moiety for energy transfer interacts with this system, energy transfer will occur. Therefore, the binary protein-protein interaction and the ternary drug-protein-protein interaction can be measured in the same experiment. Also, the fluorescent molecule only causes a signal when interacting with a protein pair, which removes any signal from the ligand interacting with an inactive protein (FIG. 149).
Example 106
6-Tetramethylrhodamine-PEG3-NH2 
To a solution of 6-Tetramethylrhodamine succinimidyl ester (0.25 g, 0.5 mmol) in DMF (5 mL), 1-Boc-4,7,10-trioxatridecan-1,13-diamine (0.15 g, 0.5 mmol) was added followed by diisopropylethylamine (0.25 mL, 1.4 mmol). After stirring for 16 h, the reaction was analyzed by HPLC to confirm complete consumption of the 6-tetramethylrhodamine succinimidyl ester. The reaction was concentrated to a pink film, which was dissolved in a combination of triisopropylsilane (0.2 mL) and trifluoroacetic acid (4 mL). The pink solution was stirred for 2 h, after which analytical HPLC confirmed complete consumption of starting material. The reaction was concentrated to dryness to provide crude 6-Tetramethylrhodamine-PEG3-NH2 as a pink film.
H-GVTGWRLCERILA-PEG-TMR (SEQ ID NO: 2578) (PBI-4877):




The fully protected peptide Boc-GVTGWRLCERILA-resin (SEQ ID NO: 2578) was synthesized by standard solid phase peptide synthesis using Fmoc techniques, then cleaved from the resin using dichloroacetic acid to liberate the fully protected peptide as a white solid. To a solution of 6-Tetramethylrhodamine-PEG3-NH2 (0.05 g, 0.08 mmol) in DMF (1.5 mL), this Boc-GVTGWRLCERILA-OH (SEQ ID NO: 2578) (0.2 g, 0.07 mmol), 1-hydroxyazabenzotriazole (11 mg, 0.08 mmol), 1-Ethyl-3-(3-dimethylaminopropyl)carbodiimide (15 mg, 0.08 mmol) and diisopropylethylamine (0.28 mL, 0.16 mmol) was added. After stirring for 30 min, the reaction was concentrated, and the resulting crude was partitioned between CH2Cl2 and water, the layers separated and the organic layer was washed with water and brine, dried over sodium sulfate and concentrated. The resulting pink solid was dissolved in a combination of triisopropylsilane (0.2 mL) and trifluoroacetic acid (4 mL). After stirring for 3 h, the reaction was concentrated, and the resulting pink film was purified with reverse phase HPLC using a gradient of ACN in 0.1% aqueous TFA to provide PBI 4877 as a pink powder: MS (M+) calcd 2088.5. found 2089.1.
TOM-DEVDGVTGWRLCERILA-OH(SEQ ID NO: 2579) (PBI-5074):




The fully protected peptide H-DEVDGVTGWRLCERILA-resin (SEQ ID NO: 2579) was synthesized by standard solid phase peptide synthesis using Fmoc techniques. While still on the resin, a solution of 6-TOM (PBI-3739) succinimidyl ester was added and allowed to react with the free N-terminus. The peptide was then cleaved from the resin and fully deprotected using trifluoroacetic acid (TFA) to provide a blue solid. This solid was purified with reverse phase HPLC using a gradient of ACN in 0.1% aqueous TFA to provide PBI 5074 as a blue powder: MS (M+Z/2) calcd 1238.9. found 1238.8.
Example 107
Complementation Comparison Between a Synthetic, N-Terminal Fusion and C-Terminal Fusion of NLPep78
Fusions of NLpep78-HaloTag (78-HT) and HaloTag-NLPep78 (HT-78) were quantitated, with a GST-HaloTag® fusion (GST-HT) as a control, by labeling E. coli lysates with the HaloTag-TMR® ligand, separated by SDS-PAGE, and scanned on Typhoon. A standard curve was then created using known concentrations of GST-HT standard, and band intensities of 78-HT and HT-78 were used to determine their concentrations.
E. coli lysates containing NLpoly11S were diluted 1:107 into PBS pH 7+0.1% Prionex. Serial dilutions of 78-HT, HT-78, and synthetic NLpep78 were made in PBS pH 7+0.1% Prionex. 20 uL NLpoly11S and 20 uL of one of the NLPep were mixed and incubated at ambient temperature for 5 minutes. 40 uL NanoGlo® reagent (Promega Corporation)+100 uM Fz were added, and the samples incubates at ambient temperature for 5 min. Luminescence was measured on GlomaxMulti+ using 0.5 s integration. Data was fit to one-site, specific binding using GraphPad Prism to determine Bmax and Kd.
The results (FIG. 150) compare the binding of NLpoly11S to synthetic NLPep78 and NLPep78 at the N- or C-terminus of a fusion partner (HaloTag). The binding affinities were not found to change significantly, but Bmax was reduced when NLPep78 was at the C-terminus.
Example 108
Complementation Comparison Between a Synthetic, N-Terminal Fusion and C-Terminal Fusion of NLPep79
Fusions of NLpep79-HaloTag (79-HT) and HaloTag-NLPep79 (HT-79) were quantitated, with a GST-HaloTag® fusion (GST-HT) as a control, by labeling E. coli lysates with the HaloTag-TMR® ligand, separated by SDS-PAGE, and scanned on Typhoon. A standard curve was then created using known concentrations of GST-HT standard, and band intensities of 79-HT and HT-79 were used to determine their concentrations.
E. coli lysates containing NLpoly11S were diluted 1:107 into PBS pH 7+0.1% Prionex. Serial dilutions of 79-HT, HT-79, and synthetic NLpep79 were made in PBS pH 7+0.1% Prionex. 20 uL NLpoly11S and 20 uL of one of the NLPep were mixed and incubated at ambient temperature for 5 minutes. 40 uL NanoGlo® reagent (Promega Corporation)+100 uM Fz were added, and the samples incubates at ambient temperature for 5 min. Luminescence was measured on GlomaxMulti+ using 0.5 s integration. Data was fit to one-site, specific binding using GraphPad Prism to determine Bmax and Kd.
The results (FIG. 151) compare the binding of NLpoly11S to synthetic NLPep79 and NLPep79 at the N- or C-terminus of a fusion partner (HaloTag). The binding affinities were not found to change significantly, but Bmax was reduced when NLPep79 was at the C-terminus.
Example 109
Spectral Scan of NLpoly11S with NLPep86 Compared to PBI-4877 (NLPep1-Fluorophore)
Purified NLpoly11S was diluted to 1 nM in PBS pH 7+0.01% Prionex+1 mM DTT. NLPep86 or PBI-4877 was diluted to 40 uM in PBS pH 7+0.01% Prionex+1 mM DTT. 25 uL NLpoly11S and 25 uL NLPep86 or PBI-4877 were mixed and then incubated at ambient temperature for 10 min. 50 uL buffer (PBS pH 7+0.01% Prionex+1 mM DTT)+100 uM Fz was then added. Luminescence was measured on Tecan Infinite M1000: 300-800 nm, every 5 nm, bandwidth 10 nm, gain 127, integration 0.5 s, z-position 22,000 um.
The results demonstrate (FIG. 152) that the NLPep can be conjugated to small molecules such as fluorescent dyes and retain interaction with NLpoly11S to produce luminescence. It also demonstrates efficient energy transfer and the ability to alter the emission spectra.
Example 110
Spectral Scan of NLpoly11S with NLPep86 Compared to PBI-5434 (Fluorophore-NLPep1)
Purified NLpoly11S was diluted to 1 nM in PBS pH 7+0.01% Prionex+1 mM DTT. NLPep86 or PBI-5434 was diluted to 40 uM in PBS pH 7+0.01% Prionex+1 mM DTT. 25 uL NLpoly11S and 25 uL NLPep86 or PBI-5434 were mixed and then incubated at ambient temperature for 10 min. 50 uL buffer (PBS pH 7+0.01% Prionex+1 mM DTT)+100 uM Fz was then added. Luminescence was measured on Tecan Infinite M1000: 300-800 nm, every 5 nm, bandwidth 10 nm, gain 127, integration 0.5 s, z-position 22,000 um.
The results demonstrate (FIG. 153) that the NLPep can be conjugated to small molecules such as fluorescent dyes and retain interaction with 11S to produce luminescence. This, along with the results with PBI-4877 in Example 109, also suggests that the terminus and/or the linker length used for the conjugation can significantly affect the energy transfer.
Example 111
Spectral Scan of NLpoly11S with NLPep86 Compared to PBI-5436 (Fluorophore-NLPep1)
Purified NLpoly11S was diluted to 1 nM in PBS pH 7+0.01% Prionex+1 mM DTT. NLPep86 or PBI-5436 was diluted to 40 uM in PBS pH 7+0.01% Prionex+1 mM DTT. 25 uL NLpoly11S and 25 uL NLPep86 or PBI-5436 were mixed and then incubated at ambient temperature for 10 min. 50 uL buffer (PBS pH 7+0.01% Prionex+1 mM DTT)+100 uM Fz was then added. Luminescence was measured on Tecan Infinite M1000: 300-800 nm, every 5 nm, bandwidth 10 nm, gain 127, integration 0.5 s, z-position 22,000 um.
The results demonstrate (FIG. 154) that the NLPep can be conjugated to small molecules such as fluorescent dyes and retain interaction with 11S to produce luminescence. It also demonstrates efficient energy transfer and the ability to alter the emission spectra.
Example 112
Comparison of Km Values for 11S with Various NLPeps in Affinity Buffer
Purified NLpoly11S was diluted to 40 pM in PBS pH 7+0.01% Prionex+1 mM DTT+0.005% Tergitol (affinity buffer) or NanoGlo assay reagent (Promega Corporation). NLPeps (NLpep86, 78, 99, 101, 104, 128 and 114) were diluted to 400 uM (NLPep to 1 mM) in affinity buffer or NanoGlo assay reagent. 300 uL NLpoly11S and 300 uL of an NLPep were mixed and incubated at ambient temperature for 30 min. 50 ul was then added to a well of white 96-well plates. 50 ul affinity buffer+2×Fz (12.5 uM diluted 2-fold 7 times) or 50 ul NanoGlo+2×Fz (100 uM diluted 2-fold 7 times) was added to each well, and luminescence measured on a Glomax Multi+ using 0.5 s integration. Km was determined using GraphPad Prism, Michaelis-Menten.
The results demonstrate substrate binding in affinity buffer (FIG. 155) or NanoGlo assay buffer (FIG. 156) to the complex between NLpoly11S and various NLPeps. The determined Km values do not fluctuate significantly with the indicated NLPeps.
Example 113
NLPep1 Binding Affinity to NLpoly11S at Various Concentrations of Furimazine
Purified NLpoly 156 and NLpoly11S to 40 pM in affinity buffer (PBS pH 7+0.01% prionex+1 mM DTT+0.005% tergitol). Synthetic NLPep1 (WT) was diluted to 560 uM for NLpoly 156 or 80 uM for NLpoly11S in affinity buffer and then serially diluted 3-fold to make 8 concentrations. 350 uL NLPep1 and 350 uL NLPoly156 or 11S were mixed and then incubated at ambient temperature for 30 min. 50 uL was then aliquoted into a well of white 96-well assay plate. Fz was added to affinity buffer to 40, 20, 10, 5, 2.5 and 1.25 uM, 50 uL Fz/affinity buffer added to each well and incubated at ambient temperature for 2 min. Luminescence was measured on a Glomax Multi+ with 0.5 s integration. GraphPad Prism and 1-site specific binding was used to calculate Kd at each concentration of Fz.
The results (FIG. 157) indicate the change in affinity (NLPoly/NLPep) with increasing concentrations of Fz.
Example 114
Furimazine Km Values for NLpoly156/NLPep1 and NLpoly11S/NLPep1 at Various Concentrations of NLPep1
Purified NLpoly 156 and NLpoly11S were diluted to 40 pM in affinity buffer (PBS pH 7+0.01% prionex+1 mM DTT+0.005% tergitol). Synthetic NLPep1 (WT) was diluted to 560 uM for NLpoly 156 or 80 uM for NLpoly11S in affinity buffer and then serially diluted 3-fold to make 8 concentrations. 50 uL was then aliquoted into a well of white 96-well assay plate. Fz was added to affinity buffer to 40, 20, 10, 5, 2.5 and 1.25 uM, 50 uL Fz/affinity buffer added to each well and incubated at ambient temperature for 2 min. Luminescence was measured on a Glomax Multi+ with 0.5 s integration. GraphPad Prism and 1-site specific binding was used to calculate Kd at each concentration of NLPep1.
The results (FIG. 158) indicate the change in affinity (NLPoly/NLPep) with increasing concentrations of NLPep1.
Example 115
Comparison of Maximal Activity for NLPoly156/NLPep1, NLPoly11S/NLPep1, and NanoLuc® Luciferase
Purified NLPoly 156, NLPoly11S, or NanoLuc® luciferase (Nluc) were diluted to 40 pM in affinity buffer (PBS pH 7+0.01% prionex+1 mM DTT+0.005% tergitol). Synthetic NLPep1 (WT) was diluted to 560 uM for NLPoly 156 or 80 uM for NLPoly11S in affinity buffer and then serially diluted 3-fold to make 8 concentrations. 350 uL NLPep1 (or affinity buffer) and 350 uL NLPoly (or Nluc) were mixed and then incubated at ambient temperature for 30 min. 50 uL was then aliquoted into a well of white 96-well assay plate. Fz was added to affinity buffer to 40, 20, 10, 5, 2.5 and 1.25 uM, 50 uL Fz/affinity buffer added to each well and incubated at ambient temperature for 2 min. Luminescence was measured on a Glomax Multi+ with 0.5 s integration. GraphPad Prism and Michaelis-Menton equation was used to calculate Vmax at each concentration of NLPep (input calculated Vmax values at each concentration of NLPep1 into 1-site specific binding to calculate Bmax). GraphPad Prism and 1-site specific binding was used to calculate Bmax at each concentration of Fz (input calculated Bmax values at each concentration of Fz into Michaelis-Menton equation to calculate Vmax).
The results (FIG. 159) demonstrate the maximal activity of NLPoly 156 or NLPoly11S upon activation by NLPep1 to the maximal activity of NanoLuc luciferase.
Example 116
Luminescent Values Resulting from Titrating NLpoly11S with Various NLPeps
Purified NLPoly11S was diluted to 40 pM in PBS pH 7+0.01% Prionex+1 mM DTT+0.005% Tergitol (affinity buffer). Synthetic NLPeps (NLPep86, 78, 79, 99, 101, 104, 114, 128 or wt) were diluted in affinity buffer as follows: NLPep86=60 nM, NLPep78=280 nM, NLPep79=800 nM, NLPep99=4 uM, NLPep101=34 uM, NLPep104=20 uM, NLPep128=4 uM, NLPep114=4.48 mM and NLPepWT=20 uM. 25 uL NLPoly11S and 25 uL an NLPep were mixed and then incubated at ambient temperature for 30 min. 50 ul affinity buffer+20 uM Fz was then added to each mixture, and luminescence measured on a GlomaxMulti+ using 0.5 s integration. Bmax and Kd values were determined using GraphPad Prism and 1 site specific binding.
The results (FIG. 160) demonstrate 100,000-fold range of affinities using NLPoly11S and various NLPeps. Minimal loss in Bmax was observed between the high affinity and low affinity NLPeps.
Example 117
Western Blot of NLPoly156, NLPoly11S, and NanoLuc® Luciferase after Transfection into HEK293T Cells
On day 1, a transfection mixture of 2 ng NLPoly156, NLPoly11S or NanoLuc® luciferase (Nluc) DNA, 1 μg pGEM3Zf(+) carrier DNA, 4 ul Fugene HD (Promega Corporation) and Phenol red-free OptiMEM to 100 ul was made and incubated at RT for 10 minutes. The transfection mixture was then transferred to one well of 6 well plate, and 2 ml of HEK293T cells at 400,000 cells/ml (800,000 cells total) was added. The cells were incubated overnight at 37° C.
On day 2, the cells were washed with phenol red-free DMEM, 500 uL phenol red-free DMEM added to each well, and the cells frozen at −70° C. for at least 30 min. The cells were then thawed, 500 uL transferred to microcentrifuge tube, and 20 ul mixed with 80 uL of 1.25×SDS loading buffer and incubated at 95° C. for 5 min. 10 ul was loaded onto 10% Bis-Tris NuPAGE gel with MES running buffer. Protein was transferred to PVDF using iBlot, and the membrane washed in methanol. The membrane was then blocked in TBST+5% BSA for 1 hr at ambient temperature, washed 3 times in TBST and then incubated with 10 mL TBST+2 uL rabbit anti-Nluc polyclonal antibody+2 uL rabbit anti-β-actin polyclonal antibody (Abcam #ab8227) at 4° C. overnight.
On day 3, the membrane was washed 3 times in TBST, incubated with 10 mL TBST+2 uL anti-rabbit HRP conjugated antibody for 1 hr at ambient temperature, washed again 3 times with TBST and incubated with 12 mL ECL Western Blotting Substrate for 1 min. Chemiluminescence was imaged with LAS 4000 Image Quant.
The results (FIG. 161) show the expression level of NLPoly compared to full-length NanoLuc® luciferase. NLPoly156 does not express as well as NanoLuc® luciferase (Nluc), whereas NLPoly11S expresses similarly to Nluc.
Example 118
Determination of the Influence of NLPoly11S/NLPep114 Affinity on the Interaction Between a β-Lactamase (SME) and β-Lactamase Inhibitory Protein (BLIP) and Comparison Between Affinity Values Measured Through 11S/114 and β-Lactamase Activity
Protein Purification
pF1K-signal-6H-SME, pF1K-signal-6H-SME-11S, pF1K-signal-6H-BLIPY50A, and pF1K-signal-6H-BLIPy50A-114 (Promega Flexi vectors for T7 promoter-based expression of recombinant protein in E. coli; the signal refers to the native signal peptide for either SME or BLIP) were induced with rhamnose to express in the periplasm of KRX cells at 25° C. for 18-20 hrs. Cells were pelleted and resuspended in B-Per lysis reagent (Pierce; 1/50th culture volume) and incubated at ambient temperature for 15 min. Lysate was then diluted by addition of 1.5× volume 20 mM Tris pH 8+500 mM NaCl and centrifuged at 12,000×g for 10 min. The supernatant was transferred to a clean tube, 1 mL RQ1 DNase (Promega Corporation) added and centrifuged again at 12,000×g for 10 min. Supernatant was purified over HisTALON column Clontech) with 25 mM Tris pH 8 and 500 mM NaCl loading buffer and eluted with 25 mM Tris pH 8, 500 mM NaCl and 50 mM imidazole. Eluted protein was dialyzed into 25 mM Tris pH 7.5 and 25 mM NaCl and purified over HiTrap Q FF column (GE Healthcare) with 25 mM Tris pH 7.5 and 25 mM NaCl loading buffer and eluted with 25 mM Tris pH 7.5 and 125 mM NaCl. Ionic strength was adjusted to final concentration of 150 mM NaCl, and concentrated using a VivaSpin concentrator.
Assay
BLIPY50A and BLIPY50A-114 were diluted to 312.5 nM in affinity buffer (PBS pH 7 0.01% prionex 0.005% tergitol 1 mM DTT), and then serially dilute 1.5-fold. SME and SME-11S were diluted to 0.2 nM in affinity buffer. 11.11 uL SME and 88.89 uL BLIP were mixed and then incubated at ambient temperature for 2 hrs. 90 uL of the mixture was transferred to a clear 96-well plate with 10 uL of 100 uM Nitrocefin (Calbiochem in affinity buffer). 90 uL of SME-11S/BLIPY50A-114 was transferred to a white 96-well plate with 10 uL of 100 uM Fz (in affinity buffer). Absorbance (nitrocefin) was measured at 486 nm every 15 sec over 30 min, and luminescence (Fz) was measure every 2 min over 30 min.
For nitrocefin, initial velocities were fit using Excel. Initial velocities vs. BLIP concentration were plotted. Fit Ki using E_free=[E]−([E_0]+[I_0]+K_app-√(([E_0]+[I_0]+K_app)^2−(4[E_0][I_0])))/2 and K_app=K_i (1+([S])/K_M) For Fz, Kd using RLU=(Bmax×[BLIP-114])/([BLIP-114]+K_D) was fit.
The results (FIG. 162) compares the affinity of a protein interaction (the β-lactamase SME and its inhibitor BLIPY50A) as unfused proteins to the affinity when NLPoly and NLPep are fused to them and demonstrates the affinity between NLPoly 11S and NLPep114 does not result in an increased apparent affinity for the SME/BLIPY50A interaction. This also demonstrates the use of NLPoly11S and NLPep114 to measure an equilibrium binding constant for a protein interaction, and the affinity measured through NLPoly11S and NLPep114 is consistent with the affinity measured by activity of the target protein (SME).
Example 119
Comparison of Luminescence Generated by Cells Expressing Different Combinations of FRB-NLPoly11S with FKBP-NLPep101 and 111-136
HEK293T cells (20,000) were reverse-transfected into wells of opaque 96-well assay plates with a total of ing pF4A Ag FRB-NLpoly11S and pF4A Ag FKBP-NLpep101 or 111-136 plasmid DNA using FuGENE HD at a DNA-to-FuGENE ratio of 1 to 8. pGEM-3Zf(+) DNA was added to bring total DNA in each transfection to 1 μg. Twenty-four hours-post transfection, cells were washed with PBS and then incubated in phenol red-free OptiMEMI with or without 50 nM rapamycin for 1.5 h. 10 μM furimazine substrate (final concentration) with or without 50 nM rapamycin in OptiMEMI was added directly to each well and incubated at room temperature for 5 min. Luminescence was then read on a GloMax Multi with 0.5 s integration time.
FIG. 163 demonstrates that, of tested combinations, NLpoly11S with NLpep114 shows the greatest rapamycin induction and one of the strongest rapamycin-specific luminescent signals.
Example 120
Comparison of Luminescence Generated by Cells Expressing Different Combinations of FRB-NLpoly11S with FKBP-NLpep114 and 137-143
HEK293T cells (20,000) were reverse-transfected into wells of opaque 96-well assay plates with a total of ing pF4A Ag FRB-NLpoly11S and pF4A Ag FKBP-NLpep114 or 137-143 plasmid DNA using FuGENE HD at a DNA-to-FuGENE ratio of 1 to 8. pGEM-3Zf(+) DNA was added to bring total DNA in each transfection to 1 μg. Twenty-four hours post transfection, cells were washed with PBS and then incubated in phenol red-free OptiMEMI with or without 50 nM rapamycin for 1.5 h. 10 μM furimazine substrate (final concentration) with or without 50 nM rapamycin in OptiMEMI was added directly to each well and incubated at room temperature for 5 min. Luminescence was then read on a GloMax Multi with 0.5 s integration time.
FIG. 164 demonstrates that, of tested combinations, NLpoly11S with NLpep114 shows the greatest rapamycin induction and one of the strongest rapamycin-specific luminescent signals.
Example 121
Rapamycin Dose Response Curves of Cells Expressing FRB-NLpoly11S and FKBP-NLpep78/79/99/101/104/114/128
HEK293T cells (20,000) were reverse-transfected into wells of opaque 96-well assay plates with a total of 0.1 ng pF4A Ag FRB-NLpoly11S and pF4A Ag FKBP-NLpep78/79/99/101/104/128 plasmid DNA using FuGENE HD at a DNA-to-FuGENE ratio of 1 to 8. pGEM-3Zf(+) DNA was added to bring total DNA in each transfection to 1 μg. Twenty-four hours post transfection, cells were washed with PBS and then incubated in phenol red-free OptiMEMI with 0 to 300 nM rapamycin for 2 h. 10 μM furimazine substrate (final concentration) with 0 to 300 nM rapamycin in OptiMEMI was added directly to each well and incubated at room temperature for 5 min. Luminescence was then read on a GloMax Multi with 0.5 s integration time. Graphpad Prism was used to fit data to sigmoidal curve and calculate EC50 values.
FIG. 165 shows a sigmoidal dose response to rapamycin for NLpoly11S with NLpep78/79/99/101/104/114/128. Of the combinations plotted, NLpoly11S with NLpep114 shows the greatest dynamic range.
Example 122
Response of Cells Expressing FRB-NLpoly11S and FKBP-78/79/99/101/104/114/128 to the Rapamycin Competitive Inhibitor FK506
HEK293T cells (20,000) were reverse-transfected into wells of opaque 96-well assay plates with a total of 0.1 ng pF4A Ag FRB-NLpoly11S and pF4A Ag FKBP-NLpep78/79/99/101/104/114/128 plasmid DNA using FuGENE HD at a DNA-to-FuGENE ratio of 1 to 8. pGEM-3Zf(+) DNA was added to bring total DNA in each transfection to 1 μg. Twenty-four hours post transfection, cells were washed with PBS and then phenol red-free OptiMEMI with 10 nM rapamycin was added for 2 h. FK506 inhibitor in OptiMEM was added to cells at final concentrations of 0 to 50 μM and incubated for 3 h. Furimazine in OptiMEM was added to cells for a final concentration of 10 μM on cells. Luminescence was immediately read on a GloMax Multi with 0.5 s integration time. Graphpad Prism was used to plot data, fit to a sigmoidal curve, and calculate IC50 values.
FIG. 166 demonstrates dose-dependent decreases in rapamycin-induced signal of FRB-NLpoly11S and FKBP-78/79/99/101/104/114/128 with the rapamycin competitive inhibitor, FK506.
Example 123
Comparison of Luminescence Generated by Cells Transfected with Different Ratios of FRB-NLpoly11S and FKBP-NLpep114
HEK293T cells (20,000) were reverse-transfected into wells of opaque 96-well assay plates with 1 ng pF4A Ag FRB-NLpoly11S and 0.01, 0.1, 1, 10, or 100 ng pF4A Ag FKBP-NLpep114 plasmid DNA using FuGENE HD at a DNA-to-FuGENE ratio of 1 to 8. HEK293T cells (20,000) were also reverse transfected with ing pF4A Ag FKBP-NLpep114 and 0.01, 0.1, 1, 10, or 100 ng pF4A Ag FRB-NLpoly11S. In both situations, pGEM-3Zf(+) DNA was added to bring total DNA in each transfection to 1 μg. Twenty-four hours post transfection, cells were washed with PBS and then incubated in phenol red-free OptiMEMI with or without 50 nM rapamycin for 1.5 h. 10 μM furimazine substrate (final concentration) with or without 50 nM rapamycin in OptiMEMI was added directly to each well and incubated at room temperature for 5 min. Luminescence was then read on a GloMax Multi with 0.5 s integration time.
FIG. 167 demonstrates that a DNA ratio of 1:1 generated the greatest rapamycin induction, although a significant induction was observed at all DNA ratios tested.
Example 124
Comparison of Luminescence Generated by Cells Expressing NLpoly11S/NLpep114 Fusions of FRB/FKBP in Different Orientations and with Different Linker Lengths
HEK293T cells (20,000) were transfected into wells of 96-well plates with vectors expressing combinations of N- and C-terminal fusions of pF4Ag NLpoly11S and pF4Ag NLpep114 with FRB or FKBP. In these constructs, NLpoly11S/NLpep114 were separated from their fusion partners with either a 4, 10, or 15 serine/glycine linker. 0.1 ng NLpoly11S and NLpep114 DNA was transfected per well at a DNA-to-FugeneHD ratio of 1 to 8. Twenty-four hours post transfection, cells were washed with PBS and then incubated in phenol red-free OptiMEMI with or without 50 nM rapamycin in OptiMEMI for 2 h. 10 μM Furimazine substrate was then added, and following a 5 min incubation at room temperature, the plate was read using a GloMax Multi with 0.5 s integration time.
FIG. 168 illustrates a rapamycin-specific increase in RLU regardless of fusion orientation or linker length.
Example 125
Comparison of Rapamycin Dose Response Curve and Time Course Generated by FRB-NLpoly11S/FKBP-NLpep114 and Split Firefly Complementation Systems
HEK293T cells (800,000) were transfected into wells of 6-well plates with a total of 20 ng pF4A Ag FRB-NLpoly11S and pF4A Ag FKBP-NLpep114 or 750 ng pF4A Ag N-Fluc(1-398)-FRB and FKBP—C-Fluc(394-544) using FuGENE HD at a DNA-to-FuGENE ratio of 1 to 4. pGEM-3Zf(+) DNA was added to bring total DNA in each transfection to 1 μg. Twenty-four hours post transfection, 20,000 cells were re-plated into wells of opaque 96-well assay plates and incubated an additional 24 h.
For dose response experiments (FIG. 169A), NLpoly11S/NLpep114-expressing cells were treated with 0-104 rapamycin in phenol red-free OptiMEMI for 3 h and then incubated with 1004 furimazine for 5 min before recording luminescence on GloMax Multi. Cells expressing N-Fluc(1-398)/C-Fluc(394-544) were incubated with 0-1 μM rapamycin in phenol red-free for 2 h, followed by an additional 1 h incubation in the presence of 4 mM D-Luciferin, prior to recording luminescence on GloMax Multi.
For time course experiments (FIG. 169B), NLpoly11S/NLpep114-expressing cells were treated with 0 or 50 nM rapamycin in phenol red-free OptiMEMI was added via GloMax Multi injector, and luminescence was immediately measured. Cells expressing N-Flu(1-398)/C-Flu(394-544) were treated with 4 mM D-luciferin in phenol red-free OptiMEMI for 1 h followed by addition of 0 or 50 nM rapamycin via injector and measurement of luminescence by GloMax Multi. Curves were fit using GraphPad Prism 6 software. FIG. 169A-B demonstrate that both NLpoly11S/NLpep114 and split firefly complementation systems respond in a rapamycin-dependent manner, generating sigmoidal dose response curves and similar EC50 values. The NLpoly11S/NLpep114 system displays faster association kinetics and a higher maximum signal.
Example 126
Comparison of FK506 Dose Response Curve and Time Course Generated by FRB-NLpoly11S/FKBP-NLpep114 and Split Firefly Complementation Systems
HEK293T cells (800,000) were transfected into wells of 6-well plates with a total of 20 ng pF4A Ag FRB-NLpoly11S and pF4A Ag FKBP-NLpep114 or 750 ng pF4A Ag N-Fluc(1-398)-FRB and FKBP—C-Fluc(394-544) using FuGENE HD at a DNA-to-FuGENE ratio of 1 to 4. pGEM-3Zf(+) DNA was added to bring total DNA in each transfection to 1 μg. Twenty-four hours post transfection, 20,000 cells were re-plated into wells of opaque 96-well assay plates and incubated an additional 24 h. Cells were then treated with 0 or 20 nM rapamycin in phenol red-free OptiMEMI for 3 h.
For FK506 dose response experiments (FIG. 170A), cells were incubated with 0 to 100 μM FK506 inhibitor in phenol red-free OptiMEMI for 5 h, treated with 10 μM furimazine, and then read with GloMax Multi in luminescence mode with 0.5 s integration time. For time course experiments (FIG. 170B), cells were treated with 10 μM FK506 in phenol red-free OptiMEMI containing 10 μM furimazine and luminescence was immediately read with GloMax Multi.
FIG. 170A-B demonstrates that the NLpoly 11S/NLpep114 and split firefly complementation systems show a dose-dependent decrease in light output following treatment with the FK506 inhibitor. The loss of signal in the NLpoly11S/NLpep114 system begins at an earlier time point, is more rapid, and is more complete than the split firefly system.
Example 127
Western Blot Showing Expression Levels of FKBP-NLpep114 and FKBP-Fluc(394-544)
HEK293T cells (200,000) were transfected with 0 to 30 ng of pF4Ag NLpep114-FKBP or pF4Ag FKBP-Fluc(394-544) DNA using FugeneHD at a DNA to Fugene ratio of 1 to 8. Forty-eight hours post-transfection, cells were harvested with 1×SDS gel loading buffer. Samples were separated on a 4-10% Tris-HCl SDS-PAGE gel and transferred to PVDF membrane. The membrane was blocked in 5% BSA in TBST for 1 h and then incubated with anti-FKBP (Abcam #ab2918) overnight. Secondary antibody incubation with horse radish peroxidase-conjugated donkey anti-rabbit IgG was performed for 1 h and then the blot was developed using ECL Western Blotting Substrate (Promega Corporation) and the Image Quant LAS 4000 system.
FIG. 171 demonstrates similar expression levels of FKBP-NLpep114 and FKBP-Fluc(394-544) at equal levels of transfected DNA.
Example 128
Dose- and Time-Specific Inhibition of NLpoly11S-BRD4 and Histone H3.3-NLpep114 Interaction by IBET-151
HEK293T cells (20,000) were transfected into wells of a 96-well white assay plate with 10 ng of pF4Ag Histone H3.3-NLpep114 and NLpoly11S-NLpoly11S using Fugene HD at a DNA to Fugene ratio of 1 to 8.
For dose response experiment (FIG. 172A), cells were treated with 0 to 10 μM IBET-151 in phenol red-free OptiMEMI for 4 h at 37° C. and then treated with 10 μM furimazine for 5 min before reading luminescence with GloMax Multi.
For time course experiment (FIG. 172B), cells were pre-incubated with 10 μM furimazine for 5 min, treated with 0-500 nM IBET-151 and immediately placed in a GloMax Multi for luminescent measurements every 5 min.
FIG. 172A-B demonstrates a dose-dependent decline in luminescence upon treatment with the BRD4 inhibitor IBET-151 that occurs within 3 hours of treatment, consistent with literature reports.
Example 129
RAS/CRAF, BRAF/BRAF, and CRAF/BRAF Dimerization in Response to GDC0879
HEK293T cells (20,000) were co-transfected into wells of 96-well assay plates with combinations of pF4Ag NLpoly 11S-BRAF, NLpoly 11S-CRAF, NLpep114-KRAS, or NLpep114-BRAF using a total of 0.1 ng DNA per well and Fugene HD at a ratio of 1 to 4. Twenty-four hours post-transfection, cells were treated with 0 to 1004 of the BRAF inhibitor GDC0879 in phenol red-free OptiMEMI for 4 h. Furimazine substrate in phenol red-free OptiMEMI was added to 1004, and luminescence was read immediately with GloMax Multi set to 0.5 s integration time.
FIG. 173 demonstrates a dose dependent increase of RAS/CRAF, BRAF/BRAF and CRAF/BRAF dimerization in response to BRAF inhibitor GDC0879.
Example 130
Twelve synthetic peptides (FIG. 180) were examined for their ability to structurally complement three different versions of NLpoly11S (i.e. 11S, 11S-amino acid 157, 11S-amino acids 156 and 157). Stocks of NLpoly were made to 35 nM in NanoGlo reagent and stocks of NLpep were made to 12.5 nM in PBS pH 7.2. Equal volumes were mixed and samples measured for luminescence on a Tecan Infinite F500 reader (100 msec integration time; 10 min time point) (FIG. 200).
Example 131
Spontaneously Interacting Peptide NLpep86
Purified NLpoly 11S was diluted to 40 pM in PBS pH 7+0.01% Prionex+1 mM DTT+0.005% Tergitol (affinity buffer). Synthetic NLPeps (NLPep86, WT, 114) were diluted in affinity buffer as follows: NLPep86=60 nM, NLPep114=4.48 mM and NLPepWT=20 uM. 25 uL NLPoly11S and 25 uL an NLPep were mixed and then incubated at ambient temperature for 30 min. 50 ul affinity buffer+20 uM Fz was then added to each mixture, and luminescence measured on a GlomaxMulti+ using 0.5 s integration. Bmax and Kd values were determined using GraphPad Prism and 1 site specific binding.
FIG. 174 demonstrates 100,000-fold range of affinities using NLPoly11S and various NLPeps. Pep 86 is an example of a spontaneously interacting peptide (with LSP 11S), and Pep 114 is shown for reference as a low affinity interacting peptide.
Example 132
Titration of High Affinity Peptide In Vitro
Purified NLpoly11S (HaloTag purification/E. coli expression; pFN18K) and synthetic peptide NLpep86 (obtained from Peptide 2.0) were titrated at a linear dynamic range using 33 nM NLpoly11S in Nano-Glo® assay buffer to 3.3 fM-100 nM high affinity NLpep86. For a 30 kDa protein, this corresponds to LOD of 10 fg.
FIG. 176 demonstrates the broad linear range and ability to detect femptamolar concentrations of the high affinity peptide tag (NLpep86). This rivals most sensitive Western Blot (WB)+Enhanced Chemiluminescence (ECL) kits
Example 133
Western Blot-Like Utility of NLpoly and NLpep
A titration of HaloTag (HT7)-NLpep 80 (80) or NLpep80-HaloTag (HT7) were run on an SDS page gel. The HaloTag® protein was imaged with HaloTag-TMR ligand (Promega Corporation) on a Typhoon scanner. The samples were transferred to a membrane and PBS pH 7+0.1% Prionex+NLpoly11S (E. coli lysate diluted 1:1,000) was used to blot the membrane. NanoGlo/Fz was then added to the membrane and it was imaged on a ImageQuant.
FIG. 177 demonstrates the sensitivity of detecting proteins tagged with a high affinity NLPep using NLpoly11S. FIG. 177 also compares the detection using NLPep/NLPoly to the detection using fluorescently labeled HaloTag.
Example 134
Stability of an NLpoly11S Reagent
100 nM NLpoly was incubated in NanoGlo assay buffer (Promega Corporation)+100 uM furimazine and assayed with equal amounts of diluted NLpep86. As a control, NanoGlo assay buffer+100 uM furimazine was used to assay an equal volume of diluted NanoLuc® luciferase (Promega Corporation).
The results (FIG. 178) demonstrate that an NLpoly reagent (containing Fz) has similar stability compared to the commercial NanoGlo® assay reagent (also containing Fz).
Example 135
Titration of DNA for High Affinity NLpep78-HT7 Fusion
HEK293 cells (200,000/ml) were reverse transfected with 10-fold dilutions of DNA (starting with 100 ng) from a high affinity peptide, NLpep78, fused to HaloTag® protein (HT7). 100 ul of each transfection was plated in triplicate into wells of a 96-well plate. Twenty-four hours post-transfection, 100 ul NanoGlo® assay buffer containing 100 nM NLpoly11S and 100 ul furimazine was added and mixed. Luminescence was measured 10 minutes after reagent addition on a GloMax luminometer.
The results (FIG. 179) demonstrate the broad linear range similar to Example 131/FIG. 27. This is essentially a similar experiment to what was done in Example 131 except that this examples uses recombinantly expressed peptide (fused to HaloTag) in a mammalian cell.
Example 136
Preliminary Results (Array Peptides)
In FIG. 183A, 50 nM NLpoly11S was mixed with 7.5 μM NLpep114 and 37.5 μM dark peptide (DP) candidate (Q-162, A-162, K-162 or E-162). NanoGlo® assay reagent (Promega Corporation) was added and incubated for 5 minutes. Luminescence was detected. In FIG. 183B, 50 nM NLpoly11S in assay buffer (PBS pH7+0.01% Prionex+1 mM DTT+0.005% Tergitol) was mixed with 7.5 μM NLpep114 (also in assay buffer) and variable amounts of dark peptide (DP) candidates Q-162 or K-162 (also in assay buffer). NanoGlo® assay reagent (Promega Corporation) was added and incubated for 5 minutes. Luminescence was detected on a Tecan Infinite F500 reader; 100 ms integration time; 5 min time point used.
Panel A indicates that each of the peptide candidates (at 7.5 uM) can inhibit the binding between NLpoly11S and NLpep114, as indicated by less bioluminescence. Note these “dark” peptides do generate some luminescence, thus the increased signal compared to no peptides at all.
Panel B indicates that with the Lys-162 and Gln-162 peptides the inhibition is dose-dependent.
Example 137
High Purity (>95%) Dark Peptides
In FIG. 184A, 5 nM NLpoly11S was mixed with 500 nM NLpep114 and variable amounts of a dark peptide (DP) candidate Q-162 or A-162 (n=3). NanoGlo® assay reagent (Promega Corporation) was added and incubated for 5 minutes. Luminescence was detected.
In FIG. 184B, 5 nM NLpoly11S in assay buffer was mixed with variable amounts of dark peptide (DP) candidates Q-162 or A-162 in assay buffer (no NLpep114)(n=3). NanoGlo® assay reagent (Promega Corporation) was added and incubated for 5 minutes. Luminescence was detected.
The results (FIGS. 184A and B) substantiate the results from Example 135, but there is greater confidence here because the peptides are more pure. These results also suggest that of the dark peptides variants tested the Ala peptide is the most potent as an inhibitor.
Example 138
Inhibition of Circularly Permuted NanoLuc® Luciferase by Dark Peptides
To determine whether “high affinity/low activity” NLpeps (a.k.a. Dark Peptides) can compete with the intramolecular interaction (i.e., protein folding) between NanoLuc® luciferase (Nluc) residues 1-156 and 157-169 in the context of circularly permuted Nluc (CP Nluc).












CP NLuc: NLuc 157-169---33aa-linker---Nluc 1-156


 








Dark peptides:
VTGWRLCERIL (SEQ ID NO: 2366) (wt)


 


1. Gln-162
VSGWQLFKKIS (SEQ ID NO: 2388)


 


2. Ala-162
VSGWALFKKIS (SEQ ID NO: 2390)









Recombinant CP Nluc was prepared as a soluble fraction of an E. coli 5×-concentrated lysate (T7-promoter; overnight expression). A 10,000-fold dilution of the CP Nluc in Assay Buffer (PBS pH 7/0.01% Prionex/1 mM DTT/0.005% Tergitol) was used. Synthetically-derived dark peptides were prepared across a range of concentrations, also in the Assay Buffer. Reactions were set up using 304 of CP NLuc and 604 of Dark peptide and assayed by adding 904 NanoGlo® assay reagent (Promega Corporation). Luminescence was measured (5 min) on a Tecan Infinite F500 reader (100 ms integration). Three replicates were used for Dark peptide samples. Two replicates were used for buffer controls (acetic acid from peptide stocks).

FIG. 185 demonstrates a dose-response of the dark peptides with CP Nluc. FIG. 186 demonstrates a time course of dark peptide (56 μM peptide) with CP Nluc.
The results indicate that both dark peptides, particularly the Ala162 version, are able to significantly inhibit generation of luminescence by CP Nluc (Ala162>2 logs; Gln162>1 log). This indicates that a CP Nluc approach has utility for inverse complementation.
Example 139
Dark Peptides in Cells
In this example, the following constructs were used:

    
    
        Four dark peptide vectors: pF4Ag+FKBP-dark peptide Ala-162, Leu-162, Gln-162 and Val-162
        Two non-dark peptide vectors: pFc5K2 FKBP-NLpep114 (low affinity peptide) and pFc5K2 FKBP-NLpep80 (high affinity peptide)
        One NLpoly vector: pFc5K2 FRB-NLpoly11S

All constructs harbored a CMV promoter for mammalian cell expression. All fusions constructs contained a 10aa Gly-Ser flexible linker.

    
    


Serial dilutions of the dark peptide constructs, Ala-162 (A), Leu-162 (L), Gln-162 (Q) and Val-162 (V), were made in OptiMem and additionally contained carrier DNA (pGEM-3Z).
For transfection containing NLpoly11S only, 20 ul of diluted dark peptide was mixed with 20 ul NLpoly11S, 60 ul OptiMem and 8 uL Fugene. For transfections containing NLpoly11S and NLpep114 or NLpep80, 20 ul of diluted dark peptide was mixed with 20 ul NLpoly11S (10 ng/ul), 20 ul NLpep114 or NLpep80 (10 ng/ul), 40 ul OptiMem and 8 ul Fugene. All were incubated at RT for 15 minutes. Sul of each transfection, in triplicate, was added to wells of two, 96-well plates (one+Rapamycin one without Rapamycin). 100 ul of HEK293T at 200,000 cells/ml in DMEM+10% FBS were then added to the wells, and the transfected cells incubated overnight at 37° C.
The medium was then removed from the cells, and the cells washed with 200 μl DPBS. 50 μl of 50 nM rapamycin was added, and the cells incubated for 1 h at 37° C. 20 μl of 5 mM furimazine in 5 ml phenol red-free OptiMEMI+50 nM rapamycin was diluted, 50 μl added directly to the cells and incubated for 5 min in GloMax Multi+. Luminescence was measured on the GloMax.
FIG. 187 demonstrates that the dark peptides, when fused to FKBP, can reduce the background signal of NLpoly11S (i.e., FRB-NLpoly11S). Taken together FIGS. 188-190 demonstrate that the dark peptides, when fused to FKBP, can 1) compete with the folding of full length NanoLuc (i.e., FRB-NanoLuc or NanoLuc-FRB) and 2) compete with both low and high affinity peptides (also FKBP fusions) for binding to NLpoly11S (i.e. FRB-NLpoly11S), and as a result reduce the total luminescence being produced and detected in live cells.
Example 140
Virology Applications
In addition to enabling measurement of viral titers, spontaneously interacting NLpeps also enable studying re-assortment of viruses (e.g., influenza). Re-assortment of viruses refers to the formation of new “hybrid” viruses from dual infections e.g. H1N1, H5N1, H3N2 (H is hemagglutinin; N is neuraminidase); bird, human, pig, chicken (most common in pigs)
Because of its segmented nature, the influenza genome can be readily shuffled in host cells infected with more than one virus. When a cell is infected with influenza viruses from different species, reassortment can result in progeny viruses that contain genes from strains that normally infect birds and genes from strains that normally infect humans, leading to the creation of new strains that have never been seen in most hosts. Moreover, because at least 16 different subtypes and nine different neuraminidase subtypes have been characterized, many different combinations of capsid proteins are possible. Of these subtypes, three subtypes of hemagglutinin (H1, H2, and H3) and two subtypes of neuraminidase (N1 and N2) have caused sustained epidemics in the human population. Birds are hosts for all influenza A subtypes and are the reservoir from which new HA subtypes are introduced into humans (Palese, 2004).
The application of the present system for detecting re-assortment is that the two components of spontaneously interacting NLpeps are be put into different viral particles, or the large component in cells and the small component in a virus, and the presence of both elements (e.g., being present in a cell) is detected by luminescence.
Example 141
Validating the Use of Spontaneously Interacting NLpep86 as an Epitope Tag for Proteins Degraded by the Proteasome
Experiments were conducted during development of embodiments of the present invention to validate the use of NLpep86 as a tag to monitor expression levels of proteins degraded by the proteasome. To do this, Nlpep86 was fused to firefly luciferase variants that were also fused to either one or more PEST, CL1 or ubiquitin sequences (pBC21, 22, 24-29). Each of these constructs is expected to undergo proteasome-mediated turnover to varying degrees following expression from a mutant CMV promoter (d1CMV).
The constructs pBC21, 22, 24-29 and control constructs expressing untagged firefly luciferase or untagged firefly luciferase fused to a PEST sequence (ATG082 and ATG083) were transiently transfected into HELA cells plated at 10,000 cells per well in a 96-well plate using 100 μL of DMEM+10% FBS. The following day, 10 μL of a transfection mixture (920 ul OptiMEM I+5 ug of the respective construct+15 ul Fugene HD) was added per well and cells were allowed to incubate for 48 hours in a 37° C. incubator containing 5% CO2. Protein expression levels were quantified in replicate wells for each construct by detecting firefly luciferase activity or by adding a detection reagent containing NLpoly 11S (purified NLpoly 11S added to NanoGlo®). A good correlation was observed between the NLpep86 and Fluc signals in each case, suggesting that NLpep86 detection can be used to monitor fusion protein expression levels for proteins degraded by the proteasome.







BC21 


(SEQ ID NO: 172) 


 


(SEQ ID NO: 2581)


MVSGWRLFKKIS-GGSGGGGSGG-Fluc (high affinity)


 


BC22 


(SEQ ID NO: 172) 


 


(SEQ ID NO: 2581)


MVSGWRLFKKIS-GGSGGGGSGG-FlucP (high affinity)


 


BC24 


(SEQ ID NO: 172) 


 


(SEQ ID NO: 2581)


pFC15A/MVSGWRLFKKIS-GGSGGGGSGG-Fluc-CL1 


 


BC25 


(SEQ ID NO: 172) 


 


(SEQ ID NO: 2581)


MVSGWRLFKKIS-GGSGGGGSGG-Fluc-PEST12opt 


(high affinity)


 


BC26 


(SEQ ID NO: 172)


 


(SEQ ID NO: 2581)


MVSGWRLFKKIS-GGSGGGGSGG-Fluc-CP 


(high affinity)


 


BC27 


(SEQ ID NO: 2583)


UBQ G76V-VGKLGRQDP-Fluc(EDAKNIKK..(SEQ ID NO: 


2582))


 


(SEQ ID NO: 2581)


GGSGGGGSGG


 


(SEQ IN NO: 390)


VSGWRLFKKIS (high affinity)


 


BC28


(SEQ ID NO: 2584)


UBQ-RGKLGRQDP -Fluc(EDAKNIKK..(SEQ ID NO: 2582))


 


(SEQ ID NO: 2581)


GGSGGGGSGG-


 


(SEQ IN NO: 390)


VSGWRLFKKIS (high affinity)


 


BC29 


(SEQ ID NO: 2585)


UBQ-LGKLGRQDP


 


 


-Fluc (EDAKNIKK..(SEQ ID NO: 2582))


 


(SEQ ID NO: 2581)


-GGSGGGGSGG 


 


-VSGWRLFKKIS (high affinity)


 


ATG083 


D1 FlucP; pF4Ag CMV Luc2-PEST


 


ATG082 


D1Fluc; pF4Ag CMV Luc2






After a 48 hour incubation, 100 uL NanoGlo® NLpep11S reagent (90 ul of NLpoly11S in 50 ml of NanoGlo® assay reagent was added to each well and incubate for 3 minutes with shaking. Luminescence was then read on GloMax luminometer (0.5 sec/well).

The results in FIG. 191 demonstrate that the signal from Fluc and NLpep86 appear to reflect each other with respect to relative brightness and have similar RLUs. BC21, BC25 and BC29 are the brightest constructs of the BC series with BC21 appearing the brightest in this experiment BC24, 26 and 27 are the least bright which is predicted from the engineered destabilization.
Example 142
This example demonstrates that a known complementing peptide can be used as a linker between the same or a different complementing NLpep and NLpoly11S (e.g., NLpep78(2×)).
HEK293T cells (20,000) were transfection with a mixture containing 20 μL of NLpep78-HaloTag (HT) or NLpep78(2×)-HT DNA, 80 μL of phenol-free Opimex and 8 μL of FugeneHD. Cells were grown overnight at 37° C. and assayed at 24 h using NanoGlo® assay reagent (Promega Corporation) containing 33 nM purified NLpoly11S.
The results (FIG. 192) demonstrate that a tandem binding peptide can be used and that it may suffice as a linker.
Example 143
Comparison of the Specific Activities of Wild-Type Oplophorus Luciferase Residues 1-156, NLpoly11S and NanoLuc in HEK293T Lysates
Each clone was inserted into pFN21A HaloTag® CMV Flexi® Vector (Promega G2821), and lysates were prepared as follows: 3 ml HEK293T cells that have been diluted to a concentration of 200,000 cells/ml (600,000 cells total) were plated into each well of a 6-well plate and grown overnight at 37° C. in a CO2 incubator. The following day transfection complexes of each DNA were prepared by combining 6.6 μg of DNA, Opti-MEM® (Life Technologies 11058-021) to a final volume of 310 μl and 20 μl of FuGENE® HD (Promega E231a). The transfection complexes were incubated for 20 minutes and then 150 μl of each complex added in duplicate to cells. The cells were grown overnight 37° C. in a CO2 incubator. The following day, the cells were washed cells with DPBS (Life Technologies 14190-144), and 1 ml fresh DPBS added. Cells were frozen to lyse and then thawed for testing. Duplicate transfection reactions lysates were combined.
To quantitate the level of protein expression for each sample, each sample was labeled with HaloTag® TMR Ligand (Promega Corporation) as follows: HaloTag® TMR Ligand (Promega G8251) was diluted 1:100 into water to a concentration of 0.05 mM; 100 μl of each lysate was mixed with 2 μl of diluted TMR ligand and incubated for 30 minutes at RT; 20 μl of SDS loading dye was added, and the samples heated to 95° C. for 5 minutes. 10 μl and 20 μl of each sample was loaded onto a polyacrylamide gel (Bio-Rad, 4-15% CriterionTM Tris-HCl Gel #345-0030.), run at 200V for 1 hour and then quantitated using ImageQuantTM LAS 4000 (GE). Both NLpoly11S and NanoLuc® luciferase (Nluc) expressed approximately 4-fold higher than 1-156.
In order to compare the specific activities of Nluc (full length enzyme) to wt Oplophorus1-156 and NLpoly11S in combination with wt Oplophorus 157-169 peptide (binary proteins), substrate titrations were run for all of the samples, but for the binary samples substrate titrations were run at multiple peptide concentrations. Using this format, it was possible to calculate a Vmax value for Nluc and both Vmax and Bmax values for NLpoly11S and wt Oplophorus 1-156. Three separate experiments were run using this format and Vmax and Bmax values were normalized to the Vmax of NanoLuc. Relative specific activities (calculated as averages of Vmax and Bmax) are normalized to NanoLuc.














Sample 
Relative specific activity









NanoLuc
1.00 



wt 1-156
0.07*



11S
0.18*







*with wt 157-169 peptide






Example 144
Effect of NLpoly and NLpep on Intracellular Half-Life of FlucP
To determine if appending either NLpoly11S or NLpep114 to Luc2-PEST alters the intracellular half-live as measured by decay of signal after cycloheximide (CHX) treatment.
Day 1: Plate Hela cells in 6 well plates. Plate 3 ml of cells (200,000/ml) into two 6 well plates. Grow overnight. DMEM+10% FBS.
Constructs containing FlucP, wt 157-169 FlucP, NLpoly11S, NLpep114 FlucP and pBC22 (all pF4Ag D1-CMV) were transfected into HeLa cells. Briefly, 33 ul of DNA (3.3 ug) was added to 122 ul of OptiMem, mixed and 9.9 ul of FuGENE® HD added. The transfection mixtures were then incubated at RT for 20 minutes, and 150 ul added to cells. After an overnight incubation, cells were replated at 10,000 cells/well and incubated again overnight.
After incubation, the growth media was removed and replaced with either 0.4 mM cycloheximide (CHX) or control (DMSO). At each time point, ONE-GloTM assay reagent was added, incubated at RT for 3 minutes and luminescence measured on Tecan GENios Pro luminometer.
FIG. 193 demonstrates that none of the NLpoly or NLpep components tested interfere with the normal intracellular degradation of a reporter enzyme (FlucP).
Example 145
Extracellular Protease Activity Assay
In some embodiments, the present invention provides an assay for extracellular protease (e.g. caspase) activity. A quenched peptide is provided (e.g., high affinity peptide such as NLpep86) that can only be accessed and refolded into an active luciferase with an NLpoly, e.g., NLpoly11S, upon removal of the quencher moiety by a protease (e.g. caspase)(FIG. 194). NLpoly11S and furimazine are introduced to the assay as a reagent and then samples are measured for bioluminescence.
Example 146
Medially Attached Pro-Groups (Isopeptides and Glycosylated Amino Acids)
Assays are provided for measuring the activity of an enzyme through using a ProNLpep. This configuration of ProNLpep is a NLpep with one of the internal amino acids conjugated to a group that prevents the complementation of the peptide to an NLpoly. When this ProNLpep encounters an enzyme that removes the blocking group (e.g., caspase 1 in the case of WEHD or a glycosidase in the case of the serine glycoside), the ability of the NLpep to complement to an NLpoly is restored (FIG. 195). In the presence of furimazine, this results in production of light in proportion to the activity of the enzyme of interest. Because each enzymatic cleavage results in the formation of a luciferase, the sensitivity of this system for assaying small concentrations of enzyme is expected to be high.
Example 147
Linker Evaluation
Assays are provided measuring the release of cargo from an antibody. An NLpep is attached to an antibody, protein, peptide, or transporter recognition moiety in such a manner that prevents it from associating with a NLpoly to form a luciferase. Upon a stimulus, such as cellular internalization, the linker between the antibody, protein, peptide, or transporter recognition moiety and the NLpep is cleaved, due to intracellular reducing potential, and the NLpep is released (FIG. 196). The NLpep can now complement with an NLpoly to form a luciferase, and the light generated will be proportional to the cleavage of the linker. This provides a system to measure the release of a compound from an antibody, which is a surrogate for cytotoxic drug delivery from Antibody Drug Conjugates. The linker can be cleaved through any manner known in the art, such as through intracellular proteases or pH sensitivity. Again, because a luciferase is generated through every cleavage, this is expected to be a sensitive method for assaying cleavage.
Example 148
The use of antibodies to target and destroy diseased cells has shown significant therapeutical promise and occurs through a process called Antibody-dependent cell-mediated cytotoxicity (ADCC). There are many ways to monitor ADCC activity, including crosslinking of different cells types or monitoring gene transcription using specific luciferase reporters expressed in the effector cells. A potential alternative readout to the ADCC mechanism of action could be in the monitoring of specific protein:protein interactions induced or disrupted after the binding of therapeutical antibodies to their target antigens or receptors presented on the cell surface. In some embodiments, the specific protein:protein interactions are monitored using the system of the present invention, which provides a readout in the time frame of minutes versus hours which is required by other methods.
Example 149
Immunoassays
Embodiments of the present invention find use in homogeneous immunoassays, for example, as depicted in FIG. 201, where the NLpep and NLpoly are fused to binding moieties (e.g., A and B). The binding moieties A and B may comprise many different components, making up several different formats of immunoassays than can be utilized as target specific assays or more generalized reagents to be used in immunoassays. The binding moieties will only come into close proximity in the presence of the target, thus bringing the NLpep and NLpoly into close proximity resulting in production of luminescence upon substrate addition. Table 7 lists exemplary of binding moieties (Mie et al. The Analyst. 2012 Mar. 7; 137(5):1085-9.
Stains et al. ACS chemical biology. 2010 Oct. 15; 5(10):943-52; Ueda et al. Journal of immunological methods. 2003 August; 279(1-2):209-18. Ueda et al. Nature biotechnology. 1996 December; 14(13):1714-8; Lim et al. Analytical chemistry. 2007 Aug. 15; 79(16):6193-200; Komiya et al. Analytical biochemistry. 2004 Apr. 15; 327(2):241-6; Shirasu et al. Analytical sciences: the international journal of the Japan Society for Analytical Chemistry. 2009 September; 25(9):1095-100; herein incorporated by reference in their entireties).










TABLE 7







Example from



(A) binding moiety
(B) Binding moiety
literature
Reference







Domain of Protein A
Domain of Protein A
Rluc fragments fused
Mie et al, Analyst,




to B-domain of
2012




protein A to detect





E. coli with primary





anti-coli Ab + fusion





complexed rabbit





anti-mouse IgG



Protein A
Protein A




Protein G
Protein G




Domain of protein G
Domain of protein G




Polyclonal Ab
Polyclonal Ab: either





same or second pAB





recognizing same





target




mAb
mAb to same target





recognizing different





epitope




scFv
scFv from an antibody
Omnitarg and
Stains et al, ACS Chem



recognizing different
Herceptin Fluc fusions
Biol, 2010; Komiya et



epitope on same
for HER2 detection; b-
al, Analytical



target
gal fusions human
Biochemistry, 2004




serum albumin





antibodies



Receptor domain 1
Receptor domain 2 to
Flt-1 domain 1 and 2
Stains et al, ACS Chem



same target
Fluc fusions for VEGF
Biol, 2010




detection



Ab variable heavy
Ab variable light chain
b-gal chain fusions
Ueda et al, J of


chain
of same antibody
for HEL; alkaline
Immunological




phosphatase and
Methids, 2003;




thioredoxine fusions
Shirasu et al,




for benzaldehyde
Analytical Sciences,





2009


Mix and match:
Mix and match:
CD4 receptor domain
Stains et al, ACS Chem


pAb, mAb, scFv,
pAb, mAb, scFv,
and scFv of anti-
Biol, 2010


receptor domain, Vh,
receptor domain, Vh,
gp120 antibody Fluc



Vl
Vl
fusions for HIV





detection;









In some embodiments in which the binding moieties are comprised of protein A, protein G, or domains of protein A or G, the immunoassay system utilizes the NLpoly and NLpep fusions to complex with antibodies prior to addition with the sample containing the target. Antibodies bind non-covalently to protein A and G naturally. Introduction of a covalent coupling between the antibody and the fusions are introduced in the complex formation step. The NLpep/NLpoly-protein A/G/domain fusions binding moieties can be complexed to the antibodies in various formats, for example:

    
    
        individually with two different specific antibodies targeting two different proteins to determine if proteins exist in complex;
        together with a single target specific polyclonal antibody;
        together with secondary antibody (e.g., rabbit anti mouse IgG) to bind to sample preincubated with primary antibody (e.g., target specific mouse IgG); and
        individually with two antibodies targeting two different epitopes on the same target protein.
    
    


As described in Table 7, in some embodiments, the binding moieties are target specific antibodies, domains of target specific antibodies, receptor domains that bind target ligands, or a combination of antibodies, antibody domains, and target receptor domains.
In some embodiments, targets are monitored in samples which include but are not limited to blood, plasma, urine, serum, cell lysates, cells (primary or cell lines), cell culture supernatant, cerebral spinal fluid, bronchial alveolar lavage, tissue biopsy samples, chemical compounds, etc.
Methods describe analysis of targets which include but are not limited to: proteins, small molecules and compounds, haptens, peptides, hormones, heterodimeric protein-protein interactions, cell surface antigens, interactions between receptors and ligands, proteins in complex, viruses and viral components, bacteria, toxins, synthetic and natural drugs, steroids, catecholamines, eicosanoids, protein phosphorylation events, etc.
Applications include but are not limited to detection or quantitation of target for clinical disease monitoring, diagnostics, therapeutic drug monitoring, biological research, pharmaceuticals, compound detection and monitoring in the food/beverage/fragrance industry, viral clade identification, etc.
Additional applications include high throughput screening of molecules capable of disrupting the interactions of target with its receptor thus resulting in a loss of signal assay. There are several proposed formats for use of NLpep/NLpoly in immunoassays. In some embodiments, these are performed homogeneously and supplied as a kit, as separate diagnostic and research kit components, or as stand-alone reagents customizable to the individual's assay.
In other embodiments, homogeneous immunoassay utilizing NLpep/NLpolyutilize variations of the HitHunter or CEDIA technology (Yang et al. Analytical biochemistry. 2005 Jan. 1; 336(1):102-7; Golla and Seethala. Journal of biomolecular screening. 2002 December; 7(6):515-25; herein incorporated by reference in their entireties). In such assays, components include: target specific antibody, NLpoly, NLpep-recombinant target fusion, and substrate. The NLpoly and NLpep-recombinant target fusion form a luminescent complex when the NLpep is not bound to the target specific antibody. Upon addition of the test sample to the assay components, the amount of luminescence is directly proportional to the target concentration in the test sample as the target present in the test sample will compete with the NLpep-recombinant target fusion on the antibody (e.g., gain of signal indicates the presence of the target).
Example 150
Exemplary Configurations for NLpoly11S in Spontaneous Complementation
Various configurations of NLpoly11S may find use in spontaneous complementation assays or systems. Such configurations may include: deletions at the C-term (e.g., to reduce background luminescence), N- and/or C-terminal appendages (e.g., based on whether they are to be purified by His or HaloTag), etc. For example, the appendage left by HaloTag when it's a N-terminal tag is SDNIAI. Exemplary configurations include: SDNAIA-11S (HaloTag purification); SDN-11S; SDNAIA-11S, with single del at C-term; SDN-11S, with single del at C-term; SDNAIA-11S, with double del at C-term; SDN-11S, with double del at C-term; SDNAIA-11S, with triple del at C-term; SDN-11S, with triple del at C-term; 6His-AIA-11S; 6His-11S; 6His-AIA-11S with single del at C-term; 6His-11S with single del at C-term; 6His-AIA-11S with double del at C-term; 6His-11S with double del at C-term; 6His-AIA-11S with triple del at C-term; 6His-11S with triple del at C-term; 11S-6His; 11S-6His, minus C-term 11S residue; 11S-6His, minus last two C-term 11S residues; 11S-6His, minus last three C-term 11S residues; 11S-HT7, minus C-term 11S residue; 11S-HT7, minus last two C-term 11S residues; 11S-HT7, minus last three C-term 11S residues; 6His-HT7-AIA-11S; 6His-HT7-11S; 6His-AIA-HT7-11S (with single, double, triple 11S C-term dels); 6His-HT7-11S (with single, double, triple 11S C-term dels); 11S-HT7-6His; 11S-HT7-6His (with single, double, triple 11S C-term dels); and Ternary 11S.
Example 151
Protein Interactions for Binary Complementation Studies
The binary complementation system described herein has been used to analyze a wide variety of protein interactions (See Table 8).







TABLE 8







Protein Interactions for Binary Complementation Studies










Interaction
Status







FRB/FKBP
Tested



V2R/ARRB2
Tested



V2R Homodimerization
Tested



BRD4/H3 3
Tested



L3MBTL3/BCLAF1
Tested



GR Homodimerization
Tested



RAS/RAF
Tested



p53/MDM2
Tested



EGFR/GRB2
Tested



BCL2/BiM/BAX
Tested



MYC/MAX
Tested



CUL1/NEDD8
In Progress



EZH2/SUZ12/EED
In Progress



GABAA Multimerization
In Progress










Example 152
Dissociation Constants and Bmax Values for NLpolys with 108 Variants of NLpeps (Array#2)
NLpeps were synthesized in array format by New England Peptide (peptides blocked at
N-terminus by acetylation and at C-terminus by amidation; peptides in arrays were synthesized at
 ̃2 mg scale) (Table 9). Each peptide was lyophilized in 2 separate plates. Each well from 1 of the plates of peptides was dissolved in 100 uL nanopure water, and the A260 measured and used to calculate the concentration using the extinction coefficient of each peptide. The concentration was then adjusted based on the purity of the peptide, and nanopure water was added to give a final concentration of 800 uM.







TABLE 9







Peptide array 2 sequences










Sequence
SEQ ID NO.





array2.1
VTGYRLFKKIS
2462


 


array2.2
VTGYRLFKKAS
2463


 


array2.3
VTGYRLFKKES
2464


 


array2.4
VTGYRLFKQIS
2465


 


array2.5
VTGYRLFKQAS
2466


 


array2.6
VTGYRLFKQES
2467


 


array2.7
VTGYRLFKEIS
2468


 


array2.8
VTGYRLFKEAS
2469


 


array2.9
VTGYRLFKEES
2470


 


array2.10
VTGYRLFQKIS
2471


 


array2.11
VTGYRLFQKAS
2472


 


array2.12
VTGYRLFQKES
2473


 


array2.13
VTGYRLFQQIS
2474


 


array2.14
VTGYRLFQQAS
2475


 


array2.15
VTGYRLFQQES
2476


 


array2.16
VTGYRLFQEIS
2477


 


array2.17
VTGYRLFQEAS
2478


 


array2.18
VTGYRLFQEES
2479


 


array2.19
VTGYRLFEKIS
2480


 


array2.20
VTGYRLFEKAS
2481


 


array2.21
VTGYRLFEKES
2482


 


array2.22
VTGYRLFEQIS
2483


 


array2.23
VTGYRLFEQAS
2484


 


array2.24
VTGYRLFEQES
2485


 


array2.25
VTGYRLFEEIS
2486


 


array2.26
VTGYRLFEEAS
2487


 


array2.27
VTGYRLFEEES
2488


 


array2.28
VTGYRLFKKIL
2489


 


array2.29
VTGYRLFKKAL
2490


 


array2.30
VTGYRLFKKEL
2491


 


array2.31
VTGYRLFKQIL
2492


 


array2.32
VTGYRLFKQAL
2493


 


array2.33
VTGYRLFKQEL
2494


 


array2.34
VTGYRLFKEIL
2495


 


array2.35
VTGYRLFKEAL
2496


 


array2.36
VTGYRLFKEEL
2497


 


array2.37
VTGYRLFQKIL
2498


 


array2.38
VTGYRLFQKAL
2499


 


array2.39
VTGYRLFQKEL
2500


 


array2.40
VTGYRLFQQIL
2501


 


array2.41
VTGYRLFQQAL
2502


 


array2.42
VTGYRLFQQEL
2503


 


array2.43
VTGYRLFQEIL
2504


 


array2.44
VTGYRLFQEAL
2505


 


array2.45
VTGYRLFQEEL
2506


 


array2.46
VTGYRLFEKIL
2507


 


array2.47
VTGYRLFEKAL
2508


 


array2.48
VTGYRLFEKEL
2509


 


array2.49
VTGYRLFEQIL
2510


 


array2.50
VTGYRLFEQAL
2511


 


array2.51
VTGYRLFEQEL
2512


 


array2.52
VTGYRLFEEIL
2513


 


array2.53
VTGYRLFEEAL
2514


 


array2.54
VTGYRLFEEEL
2515


 


array2.55
VEGYRLFKKIS
2516


 


array2.56
VEGYRLFKKAS
2517


 


array2.57
VEGYRLFKKES
2518


 


array2.58
VEGYRLFKQIS
2519


 


array2.59
VEGYRLFKQAS
2520


 


array2.60
VEGYRLFKQES
2521


 


array2.61
VEGYRLFKEIS
2522


 


array2.62
VEGYRLFKEAS
2523


 


array2.63
VEGYRLFKEES
2524


 


array2.64
VEGYRLFQKIS
2525


 


array2.65
VEGYRLFQKAS
2526


 


array2.66
VEGYRLFQKES
2527


 


array2.67
VEGYRLFQQIS
2528


 


array2.68
VEGYRLFQQAS
2529


 


array2.69
VEGYRLFQQES
2530


 


array2.70
VEGYRLFQEIS
2531


 


array2.71
VEGYRLFQEAS
2532


 


array2.72
VEGYRLFQEES
2533


 


array2.73
VEGYRLFEKIS
2534


 


array2.74
VEGYRLFEKAS
2535


 


array2.75
VEGYRLFEKES
2536


 


array2.76
VEGYRLFEQIS
2537


 


array2.77
VEGYRLFEQAS
2538


 


array2.78
VEGYRLFEQES
2539


 


array2.79
VEGYRLFEEIS
2540


 


array2.80
VEGYRLFEEAS
2541


 


array2.81
VEGYRLFEEES
2542


 


array2.82
VEGYRLFKKIL
2543


 


array2.83
VEGYRLFKKAL
2544


 


array2.84
VEGYRLFKKEL
2545


 


array2.85
VEGYRLFKQIL
2546


 


array2.86
VEGYRLFKQAL
2547


 


array2.87
VEGYRLFKQEL
2548


 


array2.88
VEGYRLFKEIL
2549


 


array2.89
VEGYRLFKEAL
2550


 


array2.90
VEGYRLFKEEL
2551


 


array2.91
VEGYRLFQKIL
2552


 


array2.92
VEGYRLFQKAL
2553


 


array2.93
VEGYRLFQKEL
2554


 


array2.94
VEGYRLFQQIL
2555


 


array2.95
VEGYRLFQQAL
2556


 


array2.96
VEGYRLFQQEL
2557


 


array2.97
VEGYRLFQEIL
2558


 


array2.98
VEGYRLFQEAL
2559


 


array2.99
VEGYRLFQEEL
2560


 


array2.100
VEGYRLFEKIL
2561


 


array2.101
VEGYRLFEKAL
2562


 


array2.102
VEGYRLFEKEL
2563


 


array2.103
VEGYRLFEQIL
2564


 


array2.104
VEGYRLFEQAL
2565


 


array2.105
VEGYRLFEQEL
2566


 


array2.106
VEGYRLFEEIL
2567


 


array2.107
VEGYRLFEEAL
2568


 


array2.108
VEGYRLFEEEL
2569









Peptides were diluted to 400 uM (4×) in PBS+0.1% Prionex and then diluted serially 7 times (8 concentrations total) in 0.5 log steps (3.162 fold dilution). NLpoly 11S was diluted 1:10^6 into PBS+0.1% Prionex. 25 uL each NLpep+25 uL NLpoly 11S were mixed and incubated for 30 min at RT. 50 uL NanoGlo+100 uM Fz was added and incubated for 30 min at RT. Luminescence was measured on a GloMax Multi+ with 0.5 sec integration. Kd/Bmax were determined using Graphpad Prism, One site-specific binding, best-fit values. Table 10 indicates the dissociation constant and Bmax values for NLpoly 11S and the indicated NLPep. The results indicate the affects of mutations on the binding to NLpoly 11S and the ability of the complex to produce luminescence.













TABLE 10






SEQ ID
Peptide






                                                                                                        
NO:
Sequence
Bmax
Kd
Bmax
Kd





















array2.1
2462
VTGYRLFKKIS
134567
0.01334
4936
0.003695


 


array2.2
2463
VTGYRLFKKAS
103904
0.2411
711.8
0.006084


 


array2.3
2464
VTGYRLFKKES
55963
0.773
1705
0.06499


 


array2.4
2465
VTGYRLFKQIS
104275
0.7462
4670
0.09318


 


array2.5
2466
VTGYRLFKQAS
31031
1.953
436.4
0.05649


 


array2.6
2467
VTGYRLFKQES
5006
1.348
182
0.1583


 


array2.7
2468
VTGYRLFKEIS
32026
4.438
1173
0.5196


 


array2.8
2469
VTGYRLFKEAS
3929
2.568
200.6
0.3566


 


array2.9
2470
VTGYRLFKEES
1453
3.863
118.6
1.044


 


array2.10
2471
VTGYRLFQKIS
112540
0.08118
4037
0.01352


 


array2.11
2472
VTGYRLFQKAS
80943
0.7485
4035
0.1039


 


array2.12
2473
VTGYRLFQKES
17237
0.4173
3190
0.3233


 


array2.13
2474
VTGYRLFQQIS
19401
0.876
2357
0.47


 


array2.14
2475
VTGYRLFQQAS
4351
1.111
311.1
0.3392


 


array2.15
2476
VTGYRLFQQES
5197
7.486
198.7
0.797


 


array2.16
2477
VTGYRLFQEIS
1321
2.561
112.6
0.5939


 


array2.17
2478
VTGYRLFQEAS
ND
ND
ND
ND


 


array2.18
2479
VTGYRLFQEES
5112
67.32
426.5
11.22


 


array2.19
2480
VTGYRLFEKIS
122961
0.6047
11827
0.2689


 


array2.20
2481
VTGYRLFEKAS
36284
1.794
935.8
0.09793


 


array2.21
2482
VTGYRLFEKES
8622
1.491
599.7
0.3267


 


array2.22
2483
VTGYRLFEQIS
121402
10.78
3711
1.121


 


array2.23
2484
VTGYRLFEQAS
3824
4.174
243.4
0.8621


 


array2.24
2485
VTGYRLFEQES
1829
7.832
24.45
0.2891


 


array2.25
2486
VTGYRLFEEIS
ND
ND
ND
ND


 


array2.26
2487
VTGYRLFEEAS
ND
ND
ND
ND


 


array2.27
2488
VTGYRLFEEES
ND
ND
ND
ND


 


array2.28
2489
VTGYRLFKKIL
140640
0.07664
6033
0.02


 


array2.29
2490
VTGYRLFKKAL
98575
0.2755
1679
0.0168


 


array2.30
2491
VTGYRLFKKEL
51143
0.6714
2000
0.07542


 


array2.31
2492
VTGYRLFKQIL
115248
2.989
2995
0.3361


 


array2.32
2493
VTGYRLFKQAL
34875
3.561
496
0.1247


 


array2.33
2494
VTGYRLFKQEL
8548
1.953
581.1
0.5209


 


array2.34
2495
VTGYRLFKEIL
21933
4.405
867.2
0.7072


 


array2.35
2496
VTGYRLFKEAL
5547
5.153
180.1
0.6609


 


array2.36
2497
VTGYRLFKEEL
1720
7.785
75.68
1.256


 


array2.37
2498
VTGYRLFQKIL
127404
0.3625
7870
0.1108


 


array2.38
2499
VTGYRLFQKAL
72788
0.9748
3853
0.1796


 


array2.39
2500
VTGYRLFQKEL
33109
2.477
687.6
0.1414


 


array2.40
2501
VTGYRLFQQIL
66256
122.3
13366
40.42


 


array2.41
2502
VTGYRLFQQAL
3472
3.97
314
1.484


 


array2.42
2503
VTGYRLFQQEL
14230
18.99
180.8
0.714


 


array2.43
2504
VTGYRLFQEIL
9406
17.25
544.2
2.141


 


array2.44
2505
VTGYRLFQEAL
4233
15.99
426.5
4.994


 


array2.45
2506
VTGYRLFQEEL
14254
35.43
614.2
3.766


 


array2.46
2507
VTGYRLFEKIL
219381
1.917
7349
0.2965


 


array2.47
2508
VTGYRLFEKAL
34526
1.807
1377
0.216


 


array2.48
2509
VTGYRLFEKEL
10865
2.437
823.9
0.5103


 


array2.49
2510
VTGYRLFEQIL
99205
124.3
2780
5.68


 


array2.50
2511
VTGYRLFEQAL
17117
40.4
294
1.642


 


array2.51
2512
VTGYRLFEQEL
46162
85
1436
4.881


 


array2.52
2513
VTGYRLFEEIL
15703
104.1
560
6.409


 


array2.53
2514
VTGYRLFEEAL
ND
ND
ND
ND


 


array2.54
2515
VTGYRLFEEEL
251166
68.27
15593
5.901


 


array2.55
2516
VEGYRLFKKIS
42384
0.07805
3011
0.02593


 


array2.56
2517
VEGYRLFKKAS
15920
0.6409
510.2
0.05975


 


array2.57
2518
VEGYRLFKKES
3374
0.891
142.5
0.1335


 


array2.58
2519
VEGYRLFKQIS
21512
2.091
665.9
0.244


 


array2.59
2520
VEGYRLFKQAS
2300
2.088
74.01
0.1938


 


array2.60
2521
VEGYRLFKQES
4346
10.64
91.51
0.7646


 


array2.61
2522
VEGYRLFKEIS
5459
14.43
116.8
0.7024


 


array2.62
2523
VEGYRLFKEAS
2375
22.05
112.3
2.964


 


array2.63
2524
VEGYRLFKEES
17264
220.3
3074
54.34


 


array2.64
2525
VEGYRLFQKIS
36517
0.5863
781.4
0.05853


 


array2.65
2526
VEGYRLFQKAS
10620
1.929
271.7
0.1454


 


array2.66
2527
VEGYRLFQKES
3489
2.87
132.3
0.3846


 


array2.67
2528
VEGYRLFQQIS
5223
8.143
199.6
0.8457


 


array2.68
2529
VEGYRLFQQAS
3753
20.01
117.8
1.833


 


array2.69
2530
VEGYRLFQQES
ND
ND
ND
ND


 


array2.70
2531
VEGYRLFQEIS
29161
230.2
560.4
6.062


 


array2.71
2532
VEGYRLFQEAS
44893
24.03
1778
1.825


 


array2.72
2533
VEGYRLFQEES
ND
ND
ND
ND


 


array2.73
2534
VEGYRLFEKIS
22544
2.148
641.3
0.2291


 


array2.74
2535
VEGYRLFEKAS
3808
4.138
122.2
0.3119


 


array2.75
2536
VEGYRLFEKES
1170
2.969
136.7
1.282


 


array2.76
2537
VEGYRLFEQIS
17957
52.79
724
4.614


 


array2.77
2538
VEGYRLFEQAS
26862
48.29
436.5
1.752


 


array2.78
2539
VEGYRLFEQES
39375
252.3
4842
41.61


 


array2.79
2540
VEGYRLFEEIS
ND
ND
ND
ND


 


array2.80
2541
VEGYRLFEEAS
383183
1419
572696
2258


 


array2.81
2542
VEGYRLFEEES
ND
ND
ND
ND


 


array2.82
2543
VEGYRLFKKIL
43371
0.563
2640
0.16


 


array2.83
2544
VEGYRLFKKAL
20849
1.588
591.2
0.1396


 


array2.84
2545
VEGYRLFKKEL
7828
1.413
ND
ND


 


array2.85
2546
VEGYRLFKQIL
31425
10.34
358.7
0.2986


 


array2.86
2547
VEGYRLFKQAL
2304
2.274
54.26
0.2428


 


array2.87
2548
VEGYRLFKQEL
1790
12.59
113
2.614


 


array2.88
2549
VEGYRLFKEIL
9831
17.75
551.8
3.002


 


array2.89
2550
VEGYRLFKEAL
5574
42.42
435.1
7.715


 


array2.90
2551
VEGYRLFKEEL
12241
100.9
458.5
6.589


 


array2.91
2552
VEGYRLFQKIL
50503
2.077
2173
0.3373


 


array2.92
2553
VEGYRLFQKAL
12294
2.023
430.5
0.206


 


array2.93
2554
VEGYRLFQKEL
4090
1.691
278.5
0.4617


 


array2.94
2555
VEGYRLFQQIL
2281
9.39
112
1.201


 


array2.95
2556
VEGYRLFQQAL
38229
18.81
1578
1.617


 


array2.96
2557
VEGYRLFQQEL
104621
99.4
6265
10.43


 


array2.97
2558
VEGYRLFQEIL
ND
ND
ND
ND


 


array2.98
2559
VEGYRLFQEAL
2696
99.9
238.8
15.53


 


array2.99
2560
VEGYRLFQEEL
ND
ND
ND
ND


 


array2.100
2561
VEGYRLFEKIL
34989
10.56
1747
1.801


 


array2.101
2562
VEGYRLFEKAL
6372
12.62
186
0.8756


 


array2.102
2563
VEGYRLFEKEL
961.5
5.786
67.06
1.216


 


array2.103
2564
VEGYRLFEQIL
ND
ND
ND
ND


 


array2.104
2565
VEGYRLFEQAL
9882
335.8
544.7
23.35


 


array2.105
2566
VEGYRLFEQEL
ND
ND
ND
ND


 


array2.106
2567
VEGYRLFEEIL
ND
ND
ND
ND


 


array2.107
2568
VEGYRLFEEAL
ND
ND
ND
ND


 


array2.108
2569
VEGYRLFEEEL
ND
ND
ND
ND









Example 153
Dark Peptides and Quencher Peptides for Reducing Background Signal from NLpoly11S
A purified sample of NLpoly11S was diluted into NanoGlo reagent to give a final concentration of 2 uM. Pep86 is a high affinity luminogenic peptide and was used to induce maximum signal for NLpoly11S. Pep86 was prepared at 1 nM in PBS (pH 7.2) for a working solution. Dark peptide and quencher peptides (FIG. 180) were dissolved to 1 mM (or lower) in either PBS pH 7.2 or 150 mM NH4HCO3 and added in equal volume to the NanoGlo/NLpoly11S and then samples were read on a Tecan Infinite F500 reader using a 5 min time point.
FIG. 202A shows that both GWALFKK (SEQ ID NO: 2351) and Dabcyl-GWALFKK (SEQ ID NO: 2351) reduce the background luminescence generated by NLpoly11S in the absence of any other luminogenic peptide. FIG. 202B shows that Pep86 is able to induce luminescence even in the presence of GWALFKK (SEQ ID NO: 2351) and Dabcyl-GWALFKK (SEQ ID NO: 2351).
FIG. 203A shows that VTGWALFEEIL (SEQ ID NO: 2372) (Trp 11mer) and VTGYALFEEIL (SEQ ID NO: 2355) (Tyr 11mer) induce luminescence over background (NLpoly11S alone; no peptide control), but that the N-terminal Dabcyl versions of each provide significant quenching of this signal. FIG. 203B shows that Pep86 is able to induce luminescence even in the presence of the Dabcyl versions of Trp 11mer and Tyr 11mer.
All publications and patents mentioned in the above specification are herein incorporated by reference. Various modifications and variations of the described method and system of the invention will be apparent to those skilled in the art without departing from the scope and spirit of the invention. Although the invention has been described in connection with specific embodiments, it should be understood that the invention as claimed should not be unduly limited to such specific embodiments. Indeed, various modifications of the described modes for carrying out the invention that are obvious to those skilled in the relevant fields are intended to be within the scope of the present invention.

## Claims

1. A system comprising
a. a cell expressing a first fusion comprising:
i. a target protein of interest, and
ii. a peptide comprising an amino acid sequence having less than 100% and greater than 40% sequence identity with SEQ ID NO: 2; and

b. a second fusion comprising:
i. an antibody binding moiety, and
ii. a polypeptide comprising an amino acid sequence having less than 100% and greater than 40% sequence identity with SEQ ID NO: 440, wherein a bioluminescent signal produced by the system in the presence of a substrate is substantially increased when the polypeptide contacts the peptide when compared to a bioluminescent signal produced in the presence of the substrate but in the absence of contact between the polypeptide and peptide.

2. The system of claim 1, wherein the target protein of interest is a membrane protein.

3. The system of claim 1, wherein the antibody binding moiety is Protein G or Protein A, Protein L, or Protein M.

4. The system of claim 1, wherein the peptide amino acid sequence is selected from the peptides of Table 1.

5. The system of claim 1, wherein the polypeptide amino acid sequence is selected from the peptides of Table 2.

6. The system of claim 1, further comprising a substrate for a bioluminescent complex produced by the peptide and polypeptide.

7. The system of claim 1, wherein the substrate is furimazine, coelenterazine, or a coelenterazine derivative.

8. The system of claim 1, wherein the second fusion further comprises a pH sensitive dye.

9. An immunoassay method comprising:
a. expressing in a cell a first fusion comprising: (i) a target protein of interest, and (ii) a peptide comprising an amino acid sequence having less than 100% and greater than 40% sequence identity with SEQ ID NO: 2, wherein a detectable bioluminescent signal is produced in the presence of a substrate when the peptide contacts a polypeptide consisting of SEQ ID NO: 440;
b. adding to the cell:
i. a second fusion comprising: (i) an antibody binding moiety, and (ii) a polypeptide comprising an amino acid sequence having less than 100% and greater than 40% sequence identity with SEQ ID NO: 440, wherein a detectable bioluminescent signal is produced in the presence of the substrate when the polypeptide contacts a peptide consisting of SEQ ID NO: 2; and
ii. a test antibody

c. adding a substrate for a bioluminescent complex produced by contact between the peptide and polypeptide, wherein said contact is mediated by binding of the antibody binding moiety to the test antibody if the test antibody binds to the target protein of interest; and
d. detecting bioluminescence generated if the antibody binds to the target protein of interest.

10. The method of claim 9, wherein the target protein of interest is a membrane protein.

11. The method of claim 9, wherein the antibody binding moiety is Protein G or Protein A, Protein L, or Protein M.

12. The method of claim 9, wherein the peptide amino acid sequence is selected from the peptides of Table 1.

13. The method of claim 9, wherein the polypeptide amino acid sequence is selected from the peptides of Table 2.

14. The method of claim 9, wherein the substrate is furimazine, coelenterazine, or a coelenterazine derivative.

15. The method of claim 9, wherein the bioluminescence detected is indicative of the concentration and affinity of the test antibody.

16. The method of claim 9, wherein the second fusion further comprises a pH sensitive dye for detection of antibody-mediated internalization.

17. The method of claim 16, further comprising detecting fluorescence if the second fusion binds to target protein of interest through an antibody and is internalized into the cell.

18. The method of claim 16, further comprising detecting BRET from bioluminescence generated when the antibody binding moiety of the second fusion binds to the antibody, which binds to target protein of interest, and is internalized into the cell.

19. A kit comprising:
a. a cell expressing a first fusion comprising: (i) a target protein of interest, and (ii) and a peptide comprising an amino acid sequence having less than 100% and greater than 40% sequence identity with SEQ ID NO: 2, wherein a detectable bioluminescent signal is produced in the presence of a substrate when the peptide contacts a polypeptide consisting of SEQ ID NO: 440;
b. a second fusion comprising: (i) an antibody binding moiety, and (ii) a polypeptide comprising an amino acid sequence having less than 100% and greater than 40% sequence identity with SEQ ID NO: 440, wherein a detectable bioluminescent signal is produced in the presence of a substrate when the polypeptide contacts a peptide consisting of SEQ ID NO: 2; and
c. a substrate for a bioluminescent complex produced by the peptide and polypeptide.

20. The kit of claim 19, wherein the target protein of interest is a membrane protein.

21. The kit of claim 19, wherein the antibody binding moiety is Protein G or Protein A, Protein L or Protein M.

22. The kit of claim 19, wherein the peptide amino acid sequence is selected from the peptides of Table 1.

23. The kit of claim 19, wherein the polypeptide amino acid sequence is selected from the peptides of Table 2.

24. The kit of claim 19, wherein the substrate is furimazine, coelenterazine, or a coelenterazine derivative.

25. The kit of claim 19, wherein the second fusion further comprises a pH sensitive dye.