# Patent

## Title
Engineered transglutaminase barrel proteins

## Abstract

Disclosed herein are methods and compositions related to engineered fragments of the human transglutaminase-related protein family, described herein as engineered transglutaminase barrel proteins (ETBPs), that have utility as high affinity, high selectivity target-binding proteins offering advantages as antibody equivalents for therapeutic, analytical, manufacturing and research purposes. ETBPs differ from naturally occurring human transglutaminase fragments by the addition, deletion, replacement and/or substitution of the naturally occurring amino acid sequence. ETBPs can be easily expressed in prokaryotic cells and in many cases can be purified by a simple solubilization and precipitation method.

## Background
CROSS-REFERENCE TO RELATED APPLICATIONS
This application is the U.S. National Stage filing under 35 U.S.C. §371 of International Application No. PCT/US2009/000016, filed Jan. 5, 2009, which claims benefit of U.S. Provisional Application No. 61/009,890, filed Jan. 3, 2008, each of which is incorporated by reference.


SEQUENCE LISTING
The instant application contains a Sequence Listing which has been submitted in ASCII format via EFS-Web and is hereby incorporated by reference in its entirety. Said ASCII copy, created on Oct. 1, 2010, is named 00786543.txt and is 266,477 bytes in size.
BACKGROUND OF THE INVENTION
In general, the present invention relates to engineered transglutaminase polypeptides, methods for their production as well as for their use for binding or for recognizing given ligands.
The adaptive immune system is a highly evolved, flexible system for the recognition and neutralization of foreign organisms and macromolecules. At the core of adaptive immunity is an engine for the creation of a vast variety of different similar structures that have been diversified by combinatorial assembly of varied building blocks with highly random linker segments. The two principle recognition complexes of the higher vertebrate adaptive immune system, antibodies and the T cell antigen receptor, are similarly assembled, and function through their cognate cell types, B cells and T cells, to effect a coordinated resistance to pathogens. Although all elements of the adaptive recognition system of higher vertebrates are based on assemblies of monomer domains of the immunoglobulin fold, in cyclostomes, convergent evolution has created an adaptive immune system that is constructed by the assembly of recognition elements derived from leucine rich repeats.
The effector proteins of the B cell arm of the adaptive immune system, particularly antibodies of the IgG subtype, have many attractive properties as candidate therapeutic agents. IgG antibodies are stable highly soluble proteins with a long in vivo half life that have weak immunogenicity within a given species. They often can be selected to have high affinities for their targets and are known to have few intrinsic safety liabilities. IgG antibodies as a class have relatively predictable behavior in vitro and in vivo, but are large, heterodimeric, disulfide-stabilized, glycosylated proteins that are difficult to make in prokaryotic cells. It has been hypothesized that antibodies may be effectively replaced for a variety of purposes by artificial antibody-like proteins, derived by the diversification of natural or unnatural scaffolds. Such antibody equivalents might be more readily manufactured and might have favorable tissue penetration and biodistribution properties compared with antibodies themselves.
In recent years recombinant antibodies of substantially human sequence have played a major role in therapeutic medicine as universal recognition moieties for a number of targets in different diseases. Human monospecific antibodies of the IgG subtype provide high specificity, bivalency, fully human composition, and long plasma half-life. The known limitations of antibodies relate largely to their biophysical properties (high molecular weight, multidomain assemblage, disulfide bonds, glycosylation), which require eukaryotic manufacturing processes that are more complex and more expensive than their prokaryotic counterparts. Fragments of antibodies, such as scFv domains, Fab domains and multivalent miniantibodies have been produced in bacteria, and offer some opportunities for the realization of low cost, highly effective therapeutic agents.
Scaffolds based on different human or non-human proteins or protein domains have emerged as an independent class of alternative therapeutic molecules. The status of alternative scaffolds and selection procedures used to identify high affinity binding proteins based on those scaffolds have been recently reviewed. Different proteins have been investigated as frameworks for bringing the diversified sequences to targets, including affibodies, lipocalins, ankyrin-repeat proteins, natural peptide binding domains, enzymes, GFP, small disulfide-bonded peptides, protease inhibitors, and others. Approximately 50 protein scaffolds have been proposed so far but only a few have been developed extensively for medical applications (Adnectins (Bristol-Myers Squibb Co), Anticalins (Pieris AG), Microbodies (Nascacell Technologies AG), Nanobodies (Ablynx), Kunitz domains (Dyax), Peptide aptamers (Aptanomics), Affibodies (Affibody AB), DARPins (Molecular Partners AG), Affilins (Scil Proteins GmbH), Tetranectins (Borean) and Avimers (Amgen)). Several are in preclinical development and a few examples are undergoing clinical trials (anti-VEGFR2 AdNectin (phase I), anti-IL6 Avimer (phase I) and engineered Kunitz-type protease inhibitor anti-kallikrein DX-88 (phase II-III)).
Although for prospective therapeutic applications to date, alternative scaffolds have largely been employed as neutralizing agents for ligand-receptor interaction, cytokine, toxin, or Fc-fusions are being investigated to confer on the binding protein a cytostatic or cytotoxic effect similar to that achieved through antibody-dependent cellular cytotoxicity (ADCC). The potential role of alternative scaffolds in diagnosis is important since large arrays of specific small reagents could be produced to many different targets. Compared to antibodies, small scaffolds should have better tissue penetration which could be advantageous for solid tumor targets.
Criteria for choosing an appropriate alternative scaffold for therapeutic purposes have been disclosed by several sources. Preferable alternative scaffolds have small size (for stability, ease of manufacturing, convenience of selection in some display methods, and tissue penetration in solid tumor applications); high thermodynamic stability and high solubility (for optimal prolonged performance in human plasma) and compatibility with therapeutic use in humans. The latter has been interpreted by some to mean that the scaffold is preferably of human origin (to avoid unwanted immunogenic effects), but scaffolds based on non-human mammalian proteins, bacterial proteins, or synthetic proteins have been proposed. Preferable scaffolds often have few disulfide bonds and free cysteines (which can lead to non-specific target binding during selection), but if the scaffold fold is stable and self-associates well in prokaryotes, as described for the type A repeats disclosed by WO 06/055689, the incorporation of cysteines may not be problematic. If the scaffold is chosen to be of human origin to minimize the adverse consequences of the generation of antibodies against the scaffold, the protein to be used as an alternative scaffold should preferably already exist in human plasma, preferably at a high concentration, and the introduction of a low titer of autoreactive antibodies to the scaffold should preferably have minimal adverse physiological consequences.
The presence of a structurally rigid core that is able to tolerate changes of surface residues without losing stability or correct folding of the protein is also desirable. Alternative scaffolds preferably exhibit protease resistance in addition to their other properties. Protease resistance can be useful for manufacturing, stability, and compatibility with biological samples or environments.
SUMMARY OF THE INVENTION
In one aspect, the invention features an optionally substituted loop-diversified and/or extension-diversified engineered transglutaminase barrel protein with at least one barrel domain derived from a Transglut-C domain of a terrestrial vertebrate (e.g., a mammalian or human Transglut-C domain).
In another aspect, the invention features a method for preparing an enriched composition of target-binding, loop-diversified or extension-diversified engineered transglutaminase barrel proteins from a terrestrial vertebrate by (i) providing a collection of nucleic acids encoding the loop-diversified or extension-diversified engineered transglutaminase barrel proteins in a display-conducive context, (ii) expressing the collection of nucleic acids in vivo or in vitro to provide a collection of loop-diversified or extension-diversified engineered transglutaminase barrel proteins operably linked to the nucleic acid that encodes them, (iii) contacting the expressed engineered transglutaminase barrel proteins with a target; (iv) removing expressed engineered transglutaminase barrel proteins that do not bind to the target; and (v) recovering the engineered transglutaminase barrel proteins enriched for binding to the target.
In another aspect, the invention features another method for preparing an enriched composition of target-binding, loop-diversified or extension-diversified engineered transglutaminase barrel proteins by: (i) providing a collection of nucleic acids encoding the loop-diversified or extension-diversified engineered transglutaminase barrel proteins, (ii) expressing the collection of nucleic acids in vivo or in vitro to provide a collection of loop-diversified or extension-diversified engineered transglutaminase barrel proteins, (iii), contacting the expressed engineered transglutaminase barrel proteins with a target; and (iv) identifying collections of expressed engineered transglutaminase barrel proteins that bind to the target.
The invention also features a method for identifying individual target-binding, loop-diversified or extension-diversified engineered transglutaminase barrel proteins by: (i) providing a collection of nucleic acids encoding the loop-diversified or extension-diversified engineered transglutaminase barrel proteins, (ii) expressing the collection of nucleic acids in vivo or in vitro to provide a collection of optionally individually indexed, loop-diversified or extension-diversified engineered transglutaminase barrel proteins, (iii) contacting the optionally individually indexed engineered transglutaminase barrel proteins with a target; and (iv) identifying collections or individual engineered transglutaminase barrel proteins that bind to the target.
In yet another aspect, the invention features an isolated nucleic acid encoding an engineered transglutaminase barrel protein or a loop-diversified and/or extension-diversified engineered transglutaminase barrel protein in an expression-conducive context.
In yet another aspect, the invention features a method for expressing and purifying a protein including at least one optionally engineered transglutaminase barrel protein domain from a prokaryotic host by: (i) expressing the protein by fermentation in the prokaryotic host; (ii) collecting the insoluble pellet resulting from lysis or disruption of the prokaryotic host under neutral to alkaline conditions; (iii) optionally washing the resulting pellet by resuspension and sedimentation; and (iv) dissolving the pellet under acidic conditions.
The invention also features a method for chemically modifying and purifying a protein including at least one optionally engineered transglutaminase barrel protein domain to achieve optimal in vivo pharmacokinetic properties of the protein by: (i) modifying the protein with a single cystein near the c-terminus that is then modified with polyethelene glycol, and (ii) purifying the modified protein by anion exchange chromatography.
In any of the foregoing aspects, the engineered transglutaminase barrel protein can be an engineered Factor XIII barrel protein.
By “branched polyalkylene glycol” is meant a branched polymer created by the joining of one or more optionally substituted oligomers or polymers of units of the form —(O—CR1R2—CR3R4)n—O—R5, where R1, R2, R3, R4, and R5, are the same or different and selected from H, F, or lower alkyl optionally substituted with one or more F; and n is 3 or greater. A branched polyalkylene glycol contains one or more linker structures (branches) of the form A-Y-linker-(X1X2) where X1═—(O—CR1R2—CR3R4)n—O—R5 and Y may be a bond or a linear polyalkylene glycol and A is a group selected to provide covalent or stable noncovalent linkage to a protein. As used, the term branched polyalkylene glycol includes block or random copolymers of units corresponding to the formula above, wherein the substituents R1, R2, R3, R4, and R5, may vary from block to block or from monomer to monomer, for example as in a block or random copolymer of polyethylene and polypropylene glycols.
By “coding sequence” is meant the sequence of nucleic acid residues that upon translation give rise to a polypeptide.
By “display” is meant any system that permits the enrichment or identification of a target-binding protein by (i) contacting a mixture of target-binding and target-nonbinding proteins each operably linked to a nucleic acid encoding said target-binding or target-nonbinding protein and (ii) separating said target-binding proteins from said target-nonbinding proteins to provide a composition enriched in the target-binding proteins compared to the initial composition.
By “display-conducive context” means any form in which a target-binding protein can be expressed such that it is available to both bind to a target and retain an operable linkage or physical relationship (for example, by association with the same host cell, phage, or fusion construct) to a nucleic acid encoding said target-binding protein.
By “engineered Factor XIII barrel protein” is meant a protein comprising one or more domains derived from the C terminal beta barrel domains of a blood-borne transglutaminase that have been modified by addition, deletion, replacement, or substitution of one or more amino acid residues.
By “engineered transglutaminase barrel protein” is meant a protein comprising one or more domains derived from the beta barrel domains of the Transglut-C family that have been modified by addition, deletion, replacement or substitution of one or more amino acid residues.
By “expressible clone” is meant a recombinant nucleic acid construct bearing an open reading frame that can be translated from N-terminus to C-terminus without termination. A “non-expressible clone” is a recombinant nucleic acid construct bearing an open reading frame that contains either frameshift or termination mutations that prevent complete translation of the coding sequence.
By “expression-conducive context” is meant the appropriate combination of flanking sequences, vector elements, regulatory sequences, or other nucleic acid sequences empirically determined to support, improve, or regulate the production of a polypeptide chain from the sequence that encodes it. The production of the polypeptide chain can be either in vitro or in vivo. For example, an expression-conducive context for a prokaryotic expression system might comprise an optionally regulated promoter of bacterial or bacteriophage origin, a ribosome binding sequence, the coding sequence of the protein to be expressed, and an optional transcriptional termination sequence. An expression-conducive context for prokaryotic in vitro expression might comprise an RNA or translatable nucleic acid comprising a ribosome binding sequence and the coding sequence of the protein to be expressed, or such expression-conducive context for prokaryotic in vitro expression might comprise a DNA or transcribable nucleic acid encoding such RNA or translatable nucleic acid as well as regulatory sequences permitting the transcription of said DNA or transcribable nucleic acid to afford said RNA or translatable nucleic acid. An expression-conducive context for eukaryotic in vitro expression might comprise an RNA or translatable nucleic acid bearing the coding sequence of the protein to be expressed and optionally bearing 5′ and 3′ flanking sequences that provide RNA stability or improve the efficiency of translation.
By “extension-diversified” as applied to a transglutaminase barrel protein is meant a transglutaminase barrel protein wherein at least one terminus, either the N-terminus or the C-terminus, of a beta barrel has been replaced and/or extended with an amino acid sequence of no greater than 30% (e.g., less than 25%, 20%, 15%, 10%, 5%, 1%, 0.1%, or less) identity to the original sequence. An extension-diversified engineered transglutaminase barrel protein may have one or both termini extended or modified.
By “Factor XIII barrel protein” is meant a protein comprising one or more domains derived from the Transglut-C family beta barrel domains of a blood-borne transglutaminase.
By “individually indexed collection” is meant any collection of species constituted as mixtures or not, so composed that the activity of any individual member can be inferred from the analysis of the activities of all mixtures.
By “isolated nucleic acid encoding a Factor XIII barrel protein in an expression-conducive context” is meant a nucleic acid composition that can be replicated in vivo or in vitro and that comprises the coding sequence for a polypeptide in an expression-conducive context, wherein said polypeptide comprises a Factor XIII barrel protein not in the native context of Factor XIII. The phrase “not in the native context of Factor XIII” means that, at minimum, the sequences encoding the catalytic domain of Factor XIII are not present.
By “isolated nucleic acid encoding a transglutaminase barrel protein in an expression-conducive context” is meant a nucleic acid composition that can be replicated in vivo or in vitro and that comprises the coding sequence for a polypeptide in an expression-conducive context, wherein said polypeptide comprises a transglutaminase barrel protein not in the native context of the transglutaminase. The phrase “not in the native context of the transglutaminase” means that, at minimum, the sequences homologous to the catalytic domain of a transglutaminase are not present.
By “loop-diversified” as applied to a transglutaminase barrel protein is meant a transglutaminase barrel protein wherein at least one loop connecting beta strands of a barrel has been replaced with an amino acid sequence of no greater than 30% identity (e.g., less than 25%, 20%, 15%, 10%, 5%, 1%, 0.1%, or less identity) to the original sequence.
By “linear polyalkylene glycol” is meant an optionally substituted oligomer or polymer of units of the form A-(O—CR1R2—CR3R4)n—O—R5, where R1, R2, R3, R4, and R5, are the same or different and selected from H, F, or lower alkyl optionally substituted with one or more F; A is a group selected to provide covalent or stable noncovalent linkage to a protein; and n is 3 or greater. As used the term linear polyalkylene glycol includes linear block or random copolymers of units corresponding to the formula above, wherein the substituents R1, R2, R3, R4, and R5, may vary from block to block or from monomer to monomer.
By “library” or “pool” is meant a collection of two or more species constituted as a single mixed entity. Preferably, a “library” or “pool” includes at least 102, 105, 1010, 1013, or 1015 members or some range within these numbers.
By “nucleic acid” is meant an optionally substituted deoxyribonucleic acid or ribonucleic acid or homologous polymer of nucleic acid bases or base analogs that can be either copied to provide an image or replica of itself or that can be translated to form a peptide, polypeptide, or protein.
By “operably linked” or “operable linkage” is meant a stable, covalent or noncovalent attachment of two or more species so described that is capable of providing a statistical association of the operably linked species sufficiently powerful that the identification or extraction of one element permits the identification or recovery of the other element in at least 10% (e.g., at least 20%, 30%, 40%, 50%, 75%, 90%, 95%, 99%, or more) of attempts.
By “peptide” is meant an optionally substituted oligomer or polymer of naturally occurring or unnatural amino acids covalently linked by one or more amide bonds.
By “stable noncovalent linkage” is meant a noncovalent association that permits the continued proximity of the two or more elements such that after a period of time encompassing the expected duration of use of the two more associated elements, no more than 50% (e.g., less than 40%, 30%, 25%, 20%, 15%, 10%, 5%, 1%, 0.1%, or less) of the elements have disassociated. For example, a stable noncovalent linkage for a complex intended to persist for 3 hours at 37° C. will have a half life of at least 3 hours at 37° C.
The term “substitution” in the context of an amino acid sequence is taken to mean the chemical or enzymatic modification of one or more amino acid residues of a polypeptide to afford a substituted polypeptide. Common forms of substitution include attachment of prosthetic groups, polymers, nucleic acids, linkers, small natural or synthetic molecules or fragments thereof, such as modifying radicals, for example methyl or other lower alkyl, formyl, acetyl, or other lower acyl, phosphoryl, or sulfonyl.
By “terrestrial vertebrate” is meant any species belonging to the taxonomic classes phylogenetically more recent than pisces, whether extant or not, including without limitation the classes mammalia, ayes, reptilia and amphibian (e.g., human).
By “Transglut-C family” is meant the family of beta barrels identified by databases of conserved protein domains, such as Pfam, family PF00927, InterPro, family IPR008958, or SuperFamily, family SSF49309.
By “transglutaminase barrel protein” is meant a protein comprising one or more domains derived from the beta barrel domains of the Transglut-C family.



BRIEF DESCRIPTION OF THE DRAWINGS
FIG. 1A is a series of schematics showing the three dimensional structures of the indicated proteins. FIG. 1A discloses SEQ ID NO: 521.
FIG. 1B is an alignment of the β-barrel 1 (SEQ ID NOS: 522-542, respectively, in order of appearance) and β-barrel 2 (SEQ ID NOS: 543-563, respectively, in order of appearance) of the protein sequences from the indicated Transglutaminase family, C-terminal domains. Seven beta sheets (A, B, C, D, E, F, and G) are underlined and highlighted in gray, α-helix is marked with a box, conserved residues are in bold, and the most conserved domain residues are marked with stars.
FIG. 2 is an alignment showing phylogeny of coagulation factor XIII beta barrel 1 and 2. The alignment of A subunit of factor XIII beta barrel domains from human (NP_000120 FXIII A) (SEQ ID NOS: 564 and 574, respectively); chimpanzee (XP_518220 FXIII) (SEQ ID NOS 565 and 575, respectively); monkey (XP_001096669 FXIII A1) (SEQ ID NOS: 566 and 576, respectively); bovine (XP_588122 FXIII A1) (SEQ ID NOS: 569 and 577, respectively); mouse (NP_083060 FXIII A1) (SEQ ID NOS: 571 and 578, respectively); rat (NP_067730 FXIII A1) (SEQ ID NOS: 570 and 579, respectively); dog (XP_857147 FXIII A) (SEQ ID NOS: 568 and 580, respectively); horse (XP_001492734 FXIII) (SEQ ID NOS: 567 and 581, respectively); opossum (XP 001368010 FXIII) (SEQ ID NOS: 572 and 582, respectively); and chicken (NP_990016 FXIII A1) (SEQ ID NOS: 573 and 583, respectively). Residues identical to human FXIII sequence are replaced with dots.
FIGS. 3A and 3B are schematic maps of the indicated expression plasmids for F13β2 scaffold. FIG. 3B discloses “His6” as SEQ ID NO: 29.
FIG. 4 is an alignment showing the nucleotide and protein sequences (SEQ ID NOS: 585, 584, and 586, respectively, in order of appearance) for wild type and optimized F13β2 scaffold. Codons optimized for E. coli and in vitro expression are highlighted in gray; single cysteine residue and T650I polymorphism are underlined.
FIG. 5A is a graph showing GFP fluorescence measured of the indicated construct under the indicated conditions. F13β2, Fn3, and mutant Fn3 (L19D, I21A, W23S) were expressed as GFP fusions in E. coli at 37° C., and GFP fluorescence ws measured at an emission of 510 nm.
FIG. 5B is an image of SDS-PAGE gels showing F13β2-GFP and F13β2-His6 “His6” disclosed as SEQ ID NO: 29) expression in E. coli under different temperature conditions as total (L) and soluble in 1×PBS, pH 7.4 (S) fractions. Non-induced BL21 cells were used as a control.
FIG. 6A is a schematic showing the structure of F13β2 scaffold as derived from the published crystal structure. Cys69 is facing toward the hydrophobic core to the domain and positioned in close proximity of other hydrophobic residues in the core (e.g., Met20). F13β2 C69V mutant was selected for construction of combinatorial libraries.
FIG. 6B is an image of SDS-PAGE gels showing expression of F13β2-C69X-GFP and F13β2-C69V-His6 (“His6” disclosed as SEQ ID NO: 29) mutant proteins as expressed in E. coli under the indicated temperature conditions.
FIG. 7 is an alignment of protein sequences of F13β2 libraries. β-strands are underlined (SEQ ID NOs:658-664); single cysteine (C69) and C69V mutation are marked with arrows; W38, y72, and randomized residues are highlighted in gray; randomized (NNS) residues are substituted with X.
FIG. 8 is a series of schematics showing the structure of F13β2 libraries. Randomized BC, DE, FG and AB, CD, EF loops are shaded black; first (Met1) residue, last (Arg102) residue, C69V mutation, and first and last residues of the randomized loops are marked on the structure; the sites of loop extensions are marked with dotted lines.
FIGS. 9A-9C are graphs showing binding of each library to beads (preclear) and target (binding) as a percent of input radioactivity. FIG. 9A is the C-NNS library, FIG. 9B is the 50% NNS library, and FIG. 9C is the A-NNS library. Myostatin concentrations used in the selections were as follows: R1 (250 nM), R2-R5 (100 nM), R6 (C-NNS and A-NNS: 100 nM; A-50% NNS: 100 and 10 nM); R7 (C-NNS and A-NNS: 100 nM and 10 nM; A-50% NNS: 10 and 1 nM), R8 (C-NNS and A-NNS: 10 nM and 1 nM; A-50% NNS: 1 nM), R9 (C-NNS: 1 nM; A-NNS: 1 and 0.1 nM). Selection pools used for further fusion production after target concentration drop are marked with an arrow.
FIG. 10 is a graph showing binding of the selected binder pool to human myostastin as measured in a radioactive equilibrium assay. The bars represent the measurements from the following samples after round 6 (A-NNS library) to M-280 beads (B); human myostatin (M100; 100 nM), protein 1 (R100; 100 nM), peptide 1 (P250; 250 nM), and protein 2 (T50; 50 nM).
FIG. 11A is a table showing the results of a radioactive binding assay for the indicated selected molecules to human myostatin. FIG. 11A discloses the “BC loop” sequences as SEQ ID NOS: 599, 599-601, 600, 599, 599, 602-603, 600, 599, 599, 599, 604, 605, 605, 605, 605, and 605, respectively, in order of appearance, the “DE loop” sequences as SEQ ID NOS: 606-607, 606, 606-607, 606-607, 607-608, 607, 606, 606, 606, 609-612, 610, and 610, respectively, in order of appearance, and the “FG loop” sequences as SEQ ID NOS: 613, 613, 613, 613-614, 614, 614, 614, 615, 615, 615-617, 617-622, respectively, in order of appearance.
FIG. 11B is a series of graphs showing binding of the indicated molecules to myostatin.
FIGS. 12A and 12B are graphs showing sample binding to myostatin under stringent conditions.
FIG. 13A is a graph showing percentage of binding between myostatin and in vitro-produced myostatin binders. E. coli-produced myostatin binder proteins disrupt the interaction between mysostatin and in vitro-produced myostatin binders. A radioactive equilibrium binding assay was performed with clones from group M1-A50 (M17), group M2-A50 (M7), and group M3-A50 (M020) (0.1 pmol) with myostatin (2 pmol) in the presence of E. coli-produced proteins from groups M1-A50 (M17), M2-A50 (M7), M3-A50 (M29), M1-A (M1-1) or wild type F13β2 domain (50 pmol). Binding was measured as a percentage of input binder material and binding in the absence of inhibiter was assigned as 100%.
FIG. 13B is a graph showing percentage of binding as a function of activin receptor IIB (ARIIB) concentration. A radioactive equilibrium binding assay was performed with clones from group M1-A50 (M17), group M2-50 (M7), group M3-50 (M29), and group M1-A (M1-1) (0.1 pmol) with myostatin (2 or 2.5 pmol) in the presence of different concentrations of ARIIB. Binding was measured as a percentage of input binder material and binding in the absence of ARIIB was assigned as 100%.
FIG. 14A is an image of an SDS-PAGE showing proteins expressed by the indicated clones in E. coli under different temperature conditions as total (L) and soluble in 1×PBS, pH 7.4 (S) fractions.
FIG. 14B upper panels are images showing fluorescence of the indicated clones of cell (C) and insoluble (I) pellets under UV light. Lower panels are images of SDS-PAGE gels showing total (L) and soluble (S) fractions of the indicated clones. Non-induced BL21 cells were used as a control.
FIG. 15 is an image of SDS-PAGE gels of proteins of the indicated groups as extracted from E. coli grown at 37° C. and extracted from the insoluble fraction. Lanes were loaded with total (L), soluble in 1×PBS pH 7.4 diluted with water (S), and extraction (E) fractions.
FIG. 16 is a graph of proliferation of MPC-11 cells as a function of hormone concentration as measured by absorbance at 450 nM using a CCK-8 kit. Squares=human activin A; triangles=human GDF-11; upside-down triangles=human myostatin.
FIG. 17A-17D are graphs showing inhibition of cell proliferation as a function of indicated myostatin binding protein concentration. Blocking of myostatin-induced inhibition of cell proliferation was measured by absorbance at 450 nM using CCK-8 kit, with absorbance of growth media subtracted, and absorbance of cells untreated with myostatin assigned as 100%. Myostatin, used at a concentration of 125 ng/mL inhibited cell proliferation to approximately 20% of untreated cells. Values are means+/−S.D., n=3 replicates.
FIG. 18A is a schematic showing the structure of Y-Mal-40K (Y-Shape PEG Maleimide, MW 40 kDa; JenKem Technology USA Inc., Allen, Tex.).
FIG. 18B is an image of an SDS-PAGE gel showing three pegylated forms of F13β2 myostatin binder F10. Tagless myostatin binding protein F10 (PCM) was expressed in E. coli at 37° C., purified from insoluble fraction and pegylated with Y-MAL-40K.
FIG. 19A is a schematic showing the configuration of E3 homodimer molecule for expression. FIG. 19A discloses “His6” as SEQ ID NO: 29.
FIG. 19B is an image of SDS-PAGE gels showing expression of E3 homodimers in E. coli. Proteins were purified from the insoluble fraction. Lanes were loaded as described above.
FIG. 20 is a graph showing binding of A-50% NNS library to human β-NGF. Binding to the beads (preclear) and to the target (binding) was measured as a percentage of input radioactivity. Human β-NGF concentrations used in the selections were as follows: R1 (250 nM), R2-R6 (100 nM), R7-R8 (10 nM), R9 (1 nM). Selection pools used for further fusion production after target concentration drop are marked with an arrow.
FIG. 21A is a table showing characterization of binding affinities of selected molecules to human β-NGF. Binding affinities were measured in a radioactive equilibrium binding assay. FIG. 21A discloses the “BC loop” sequences as SEQ ID NOS: 623-628, the “DE loop” sequences as SEQ ID NOS: 629-634 and the “FG loop” sequences as SEQ ID NOS: 635-640, all respectively, in order of appearance.
FIG. 21B is a graph showing binding as a percentage of input of the indicated construct to the indicated concentration of NGF.
FIG. 22A is a graph showing binding of Aext-NNS library as a percentage of input to beads (Preclear) and target (Binding). Human FcεRI concentrations used in the selections were as follows: R1 (100 nM), R2 (50 nM), R3 (25 nM), R4 (12.5 nM), R5 (6.25 nM), R6 3.125 nM), R7 (16 and 1.6 nM), R8 (0.1 nM). Selections pools used for further fusion production after target concentration drop are marked with arrows.
FIG. 22B is a graph showing binding of the library (R0) and of free protein as a percentage of input after each rond (PR1-5, 7, 8) to the beads and to the target (50 nM) as measured in a radioactive equilibrium binding assay.
FIG. 23 is a graph and table showing binding as a percentage of input of indicated binding pools to human FcεRI.
FIGS. 24A and 24B are schematics showing maps of expression plasmids for F13β1 scaffold and F13β1β2 heterodimer. FIGS. 24A and B disclose “His6” as SEQ ID NO: 29.
FIG. 25 is an alignment showing the nucleic acid (SEQ ID NO: 641) and protein (SEQ ID NO: 642) sequences of wild type F13β1 scaffold.
FIGS. 26A and 26B are images of SDS-PAGE gels showing expression of the indicated proteins at the indicated concentrations. FIGS. 26A and B disclose “His6” as SEQ ID NO: 29.
FIGS. 27A and 27B are schematics of the proposed F13β1 (27A) and F13β1β2 (27B) libraries. Potential randomization in BC, DE, FG, and AB, CD, EF loops are colored in black.
FIG. 28 is an alignment showing the protein sequences (SEQ ID NOS: 643-648, respectively, in order of appearance) of the proposed F13β1 libraries. B-strands are underlined; randomized (NNS) residues are highlighted in gray and substituted with X.
FIG. 29 is a schematic showing representations of ETBP and M13 pIII fusions under lacZ promoter control. The fusion DNA was constructed by an overlapping PCR strategy. The resulting constructs encode translational fusions of FXIIIβ2 or myostatin-binding clone E3 fused to either mature full length M13 pIII or the C-terminal domain of M13 pIII. FIG. 29 discloses “His6” as SEQ ID NO: 29.
FIGS. 30A and 30B are a schematics showing DNA maps of FXIIIβ2 placed under the lacZ promoter (30A) or pBAD promoter (30B) control. FIG. 30A discloses “His6” as SEQ ID NO: 29.
FIG. 31A is a graph showing ETBP displaying phage binding to myostatin in an ELISA assay. Four phage preparations are indicated: FXIIIβ2pIII (square), FXIIIβ2pIIICT (triangle), E3-pIII (upside-down triangle), and E3-pIIICT (diamond). Bound phage was detected with an anti-m13 pVIII antibody conjugated to HRP.
FIG. 31B is a graph showing phage recovery after binding to myostatin. Four phage preparations were bound to myostatin coated wells. Bound phage was eluted with a low pH solution (gray) or used to infect E. coli TG1 cells in wells directly (white). Recovered phage number were determined by ampicilin resistant colony forming units.
FIGS. 32A and 32B are graphs showing ETBP displaying phage binding specificity. Phage prepartions from E3-pIII (32A) and E3-pIIICT (32B) were tested for binding specificity in ELISA. The following compounds were immobilized on wells of Maxisorb plates and reacted with phage: myostatin (squares), myostatin-biotin (triangles), mbpFcεRI (upside-down triangles), and BSA (diamonds). Bound phage was detected with an anti-M13 pVIII antibody conjugated to HRP.
FIGS. 33A and 33B are graphs showing quantity of ETBP displaying phage propagated in the presence of glucose as measured by ELISA. E3-pIII and E3-pIIICT phage were propagated in 2XYT supplemented with different amounts of glucose: 0% (squares), 0.02% (triangles), 0.2% (upside-down triangles), and 2% (diamonds). Phage was reacted with myostatin coated wells.
FIG. 34 is an image of a Western blot showing ETBP displaying phage. E3-pIII and E3-pIIICT phage were propagated in 2XYT supplemented with different amounts. The membrane was first probed with an anti-pIII antibody (MoTec) followed by a goat anti-mouse Fc antibody conjugated to HRP.
FIG. 35 is a graph showing ETBP phage propagated in the presence or absence of arabinose as measured using ELISA. paracompE3pIII phage was propagated in 2XYT supplemented with: 0 mg/mL (squares), 0.01 mg/mL (triangles), 0.1 mg/mL (upside-down triangles), or 1 mg/mL arabinose. Phage was reacted with myostatin coated wells. Bound phage was detected with an anti-M13 pVIII antibody conjugated to HRP.
FIG. 36 is a schematic showing diversification of ETBP phage display library. The A-FGext library randomization strategy is shown. Library inserts were cloned into BbsI digested E3BbsIpIIICT. The resulting library has a sequence diversity of 5×108. FIG. 36 discloses SEQ ID NOS: 29 and 649-651, respectively, in order of appearance.
FIG. 37 is a schematic showing the randomization strategy for FG6 and FG6+6 libraries. Randomized positions are underlined in larger font. Oligonucleotides used for randomization are shown. For each position, 50% of wildtype residues are preserved. Exact composition of each randomized position is shown. FIG. 37 discloses SEQ ID NOS: 653, 652, and 654-657, respectively, in order of appearance.
FIG. 38 is a series of graphs showing phage recovery from FG6 and FG6+6 selections to FcεRI and lysozyme. Total phage number was determined by tittering a fraction of elution from each round of selection. Elution from both target-coated and control wells were titered. Target and library used in each selection is shown in graph title.
FIG. 39 is a series of graphs showing selected clones binding to targets in phage ELISA. Randomly picked clones were analyzed after the fourth round of selection. Phage was incubated with Maxisorp wells coated with or without target. Binding level was determined using an anti-M13 antibody conjugated to HRP.
FIGS. 40A and 40B are a series of graphs showing the pharmacokinetics of PEGylated myostatin ETBPs. PEGylated monomeric (FIG. 40A) and tandem dimeric (FIG. 40B) myostatin binder were injected subcutaneously to FBV mice at 5 mg/kg. Plasma samples were collected at indicated time points post injection. Binder concentration was determined by ELISA. Pharmacokinetic modeling was performed using the WinNonLin software (Pharsight).



DETAILED DESCRIPTION OF THE INVENTION
Members of the transglutaminase family of proteins share a common four domain structure consisting of a β-sandwich domain, core domain, and β-barrel 1 and β-barrel 2 domains, first identified in the structure of the coagulation factor XIII A1 polypeptide (Yee et al. Proc. Natl. Acad. Sci. USA 1994, 91:7296). The transglutaminase family of proteins falls within a larger transglutaminase superfamily that includes proteins that share homology to the core domain but often lack the C-terminal barrel structures, and can act as proteases or peptide N-glycanases instead of transglutaminases. In the PFAM conserved domain database, the individual β-barrels of the transglutaminase family are classified as comprising the Transglut-C domain family (pfam000927). Human proteins that include β-barrel domains belonging to the Transglut-C domain family presently comprise Factor XIII A1 chain, seven proteins identified as transglutaminase-1 through transglutaminase-7, the erythrocyte membrane protein band 4.2, the protein identified by GenPept/SwissProt record Q6ZTL3, and the protein identified by GenPept/SwissProt record Q9H035.
Transglutaminases vary greatly in sequence between and within species, but share the conserved structural domains, including the beta barrels (FIGS. 1A and 1B). For example, the fish TGase has only 29% sequence identity with human FXIII in the barrel 2 domain, but has a structure very similar to human FXIII, including all four sequential domains in a conserved spatial orientation (Noguchi et al. J. Biol. Chem. 2001, 276:12055) (FIG. 1A).
The two C-terminal domains have a seven-stranded β-barrel fold and “3+4” sheet structure (FIG. 1B). They each contain approximately 100 residues, are 50 Å long and 25 Å wide. In Factor XIII, the β-barrel 1 sequence extends from Ser517 to Leu628 and the β-barrel 2 sequence extends from Thr629 to Met732. The choice of starting point for the numbering of the beta barrel sequences is to some extent arbitrary, and from the crystallographic coordinates the core sequences that comprise the termini of the strands of the second barrel spans Ile633 to Arg728. The residues from Leu628 to Ile633, exclusive, can be considered linker sequences.
The 7 beta strands of barrels of the family are designated strands A through G (FIG. 1B). The A, C, E and G strands extend in the same approximate direction and the B, D, and F strands in the opposite direction. The overall organization of the barrel, with the N-terminus prior to the beginning of the A strand and the C-terminus at the end of the G strand, establishes a natural polarity of the barrel in the direction of the A, C, E and G strands. The strands are connected to one another by loops, of which AB, CD, and EF are located at the end proximate to the C terminus of the barrel, and BC, DE, and FG are located at the end proximate to the N-terminus of the barrel.
Alternative scaffolds drafted as domains from larger proteins preferably have a compact, autonomous structure that is stable in the absence of the remaining domains of the protein. The Transglut-C domains that have been studied to date appear to possess this attribute. For example, both C-terminal domains of the human FXIII-A molecule have been reported to be stable at extremely high temperatures (Tm=90-110° C.) and to exhibit independent folding, as demonstrated by spectroscopic methods.
Although the two barrel domains of transglutaminases are structurally related, there is usually no significant primary sequence similarity between them, nor is their similarity in sequence to fibronectin type III domains. An examination of the two barrel domains reveals that their conserved and diverse sequence segments are similarly arranged, with most variations between BC and CD loops, CD and DE loops, and EF and FG loops (FIG. 1B). Several nearly invariant residues, including Asn541, Asn672, Gly562, Gly671, Pro579, and Pro685, are spatially located in the loop regions: Asn541 and Asn672 in BC loop, Gly562 and Gly671 in CD loop, and Pro579 and Pro685 in DE loop.
Among human transglutaminases that might be used as a source for diversified β-barrel proteins, coagulation factor XIII is of particular interest because it is naturally found in plasma. Proteins that are naturally found in plasma may be better templates for diversification of proteins that are intended to be used therapeutically. For example, auto-immune reactions against plasma proteins induced by cross-reactivity to a therapeutically administered protein may have less severe consequences than auto-immune reactions directed against intracellular proteins as the latter reactions might destroy cells, tissues or organs, or have other deleterious consequences. For example, the autoimmune diseases dermatitis herpetiformis and celiac sprue are known to be accompanied by autoantibodies against epidermal transglutaminase (transglutaminase 3) and tissue transglutaminase (transglutaminase 2). β-barrel domains from transglutaminase family proteins expressed only or predominantly in immunologically privileged cells may also have reduced capacity to induce autoimmune syndromes if administered therapeutically. An example of such a transglutaminase family protein is erythrocyte band 4.2 protein.
Factor XIII (FXIII) is the final zymogen of the blood coagulation cascade of higher vertebrates, and is responsible for the crosslinking of fibrin peptides to form mechanically and enzymatically stable clots. In blood platelets, megakaryocytes, monocytes, macrophages, spleen, chondrocytes and placenta FXIII is found as a dimer of identical catalytic A subunits (83 kDa), and in plasma as a heterotetramer of two A and two noncatalytic B subunits (90 kDa) noncovalently linked together and bound to fibrinogen. The carrier B subunit, normally present in excess in plasma, stabilizes the A subunit and protects it from proteolysis. FXIII is not secreted by the classical secretory leader-dependent pathway, but exits the cell by a poorly understood mechanism.
The A subunit of FXIII is activated by thrombin cleavage C-terminal to residue 37 of the protein (Takagi and Doolittle Biochemistry 1974, 13:750). In the presence of Ca2+, activated plasma FXIII dissociates from its noncatalytic B subunits; the A subunits undergo a conformational change to assume the enzymatically active form FXIIIa, which exercises its transglutaminase activity on fibrin provided by the action of thrombin (Factor II) on fibrinogen. Fibrin itself promotes the activation of FXIII by serving as a cofactor for the calcium-dependent conformational change of FXIII-A, which in turn allows the thrombin catalyzed activation of FXIII to FXIIIa. In the absence of Ca2+ and at high thrombin concentrations a second thrombin cleavage can also occur on the 79-kDa enzyme leading to the formation of 25- and 54-kDa fragments. FXIIIa forms intermolecular γ-glutamyl-ε-lysylamide crosslinks between noncovalently polymerized fibrin macromolecular complexes, fibrin monomers, α-2 antiplasmin, fibrinogen, fibronectin, collagen, and other proteins to enhance the mechanical strength of the fibrin clot, protect it from proteolytic degradation, and provide stability to the extracellular matrix.
The average concentration of FXIII in human plasma is approximately 150 nM. Rare cases of factor XIII A chain deficiency result in inefficient wound and bone healing, severe bleeding, high risk for spontaneous abortion and intracranial hemorrhage, and are treated by substitution therapy. Exogenously administered FXIII has a half-life of 9-10 days.
Although there is no direct evidence of involvement of β-barrels in the enzymatic function of transglutaminase family proteins, the movement of the β-barrel region upon activation has been proposed and/or demonstrated. For example upon binding to an active site inhibitor, transglutaminase 2 undergoes a profound change in conformation in which the β-barrels are rotated away from the catalytic core, forming a highly extended structure (Pinkas et al., PLoS Biol. 2007, 5:e327). Similarly, in Factor XIII, Tyr560, positioned on a loop of β-barrel 1, is involved in hydrogen-bonding with the active site cysteine of FXIII (Cys314) and is probably displaced by the glutamine donor substrate which approaches the active site from the direction of the two β-barrel domains. Hydrogen/deuterium exchange (HDX) studies have shown that Ca2+ binding could potentially direct β barrel 1 to roll away from the catalytic core to allow glutamine substrate access (Sabo et al, Biochemistry 2007, 46:10089). Within β-barrel 2, chemical modification and proteolysis studies have provided evidence for C695 exposure upon activation. It has been shown that glutamine-bearing substrates create a more solvent accessible region within β-barrel 2, which in turn is potentially involved in lysine substrate recognition. This hypothesis is supported by the observation that antibody 5A2, which targets the FXIIIa epitope 646-658, displays uncompetitive inhibition toward the glutamine substrate and competitive inhibition against the lysine substrate.
Factor XIII-related proteins found in different species appear to be highly conserved in length and sequence. The beta barrel domains within these proteins are highly conserved (FIG. 2). For example, the chicken factor XIII polypeptide has 55% sequence identity to the human polypeptide in the beta barrel region (from Asn518 to the Arg728) whereas the rhesus macaque factor XIII polypeptide shares 94% sequence identity from Val519 to Met732.
Functional recombinant A subunit of FXIII has been produced in yeast and the recombinant product has entered clinical studies for treatment of FXIII-A deficiency conditions. Conflicting data have been disclosed regarding E. coli expression of FXIII-A: one study reported only non-functional recombinant FXIII-A, while another reported expression of fully functional FXIII-A with properties similar to native plasma or yeast-produced FXIII-A chains. To date the production of individual beta barrel domains of FXIII by recombinant methods has not been disclosed, although highly stable 24 kDa and 12 kDa C-terminal fragments, corresponding to β1β2 and β2 barrels have been obtained by elastase digestion from recombinant FXIII-A.
The present invention provides compositions based on beta barrel domains of transglutaminases, described herein as engineered transglutaminase barrel proteins (ETBPs). ETBPs comprise one or more artificially diversified transglutaminase beta barrel domains. An ETBP may be solely comprised of barrel one domains or solely comprised of barrel two domains or may be comprised of mixtures of the two barrel types. In particular an ETBP may contain diversified examples of both barrels in the native organization in which they are found in Factor XIII, erythrocyte band 4.2 protein, or the tissue transglutaminases 1-7, in which barrel one lies N terminal to barrel two. ETBPs are derived from naturally occurring barrel sequences by the addition, deletion, or substitution of residues in the naturally occurring barrel sequences. Of particular interest are the ETBPs that have been derived by substitution of one or more loops of a naturally occurring barrel sequence, or that have been derived by the addition of one or more peptide extensions at the N-terminus and/or the C-terminus. ETBPs derived by modification of loop sequences are called loop-diversified ETBPs whereas ETBPs derived by extension are called extension-diversified ETBPs. A given ETBP may be both loop-diversified and extension-diversified, and may contain additional sequence variation, for example improving affinity, stability, selectivity, or solubility, that have been introduced at any location in the molecule. In addition an ETBP may be optionally substituted with prosthetic groups, polymers, proteins, nucleic acids, carbohydrates, metals, or natural or synthetic small molecules.
Diversity of loop or extension regions in ETBPs is introduced through the incorporation of diversity in nucleic acids encoding ETBPs. For this purpose random or indexed collections of nucleic acids encoding different sequences can be prepared according to any of several methods known in the art. Such collections can be prepared in ways that favor specific sequences or residues, or disfavor specific sequences or residues, by altering the probability of appearance of specific nucleotides in a site-specific or site-non-specific manner, or by specifying triplets of nucleic acid residues corresponding to individual codons and varying the relative abundance of said triplets in a site-specific or site-non-specific manner. For example, to reduce the abundance of cysteine residues, encoded in DNA by TGY, the relative abundance of T in the first position, G in the second position, or a pyrimidine in the third position, can be reduced in the template nucleic acid, illustrated here as DNA. Alternatively a mix of triplet precursors corresponding to each of the individual amino acids could be prepared, from which the triplets TGT and TGC are excluded. To reduce the frequency of termination codons a common strategy is to randomize loops or extensions in the form (NNS)n, where n denotes the number of random triplets to be included, N represents any nucleotide, and S represents C or G in the standard IUPAC nucleotide nomenclature. Since two of three stop codons (TGA and TAA) have an A in the third position, an NNS strategy is expected to reduce the frequency of stop codons from 3/64 to 1/32 compared to an NNN strategy.
The preparation of loop-diversified ETBPs may be carried out by any of several different schemes well known in the art. For example, libraries of ETBPs containing loop regions replaced by random peptide sequences of the same or different length can be prepared by recombinant DNA methods. Such libraries can be prepared as variants of a single scaffold having up to six loops diversified in a single ETBP or can be prepared as separate libraries each having one diversified loop per ETBP, or by some combination of loop-diversified libraries, for example having two diversified loops at one end and one at the other. Schemes in which individual libraries of loops are provided are often coupled with a facility for the interchange of the individual libraries, for example by the placement of restriction enzyme cleavage sites in the non-diversified regions that permit shuffling and reassembly of the library templates by restriction enzyme digestion and ligation. Other methods for shuffling of existing sequences are well-known in the art, and include stimulated recombination in vitro or in vivo, and nucleic acid shuffling and rejoining by PCR or isothermal amplification. In some cases it may be useful to create sublibraries from existing libraries, or sublibraries that incorporate some previously selected candidate ETBP as a starting point for the generation of further diversity focused on a particular ETBP with desirable properties. For example, to further improve the affinity of a pool of candidate ETBPs that shows a predominance of one loop sequence, a new library can be created in which that loop sequence is taken as the starting point and further randomization is introduced by partial or complete randomization of the other loops and/or by partial randomization of the predominant loop.
Extension randomization can be similarly carried out at the N-terminus or C-terminus or both. The starting point for randomization can be either a naturally occurring transglutaminase barrel or a preselected candidate ETBP. The extension-diversified ETBP is created by the addition of one or more residues to the N-terminus or C-terminus or both. Very long extensions are likely to compromise the stability or solubility of the ETBP and hence practical extensions are in most cases likely to be less than fifty residues in length, more preferably less than forty residues in length, and most preferably less than 20 residues in length, although longer extensions can be formed by intermediate selection for ETBP starting points that have favorable stability or solubility. Further randomization of an initially preselected extension-diversified ETBP can be focused on a preselected sequence by introducing, through the use of diversified nucleic acid templates, a weighted probability of mutation at each residue that is provided to introduce a predetermined number of changes per extension. The weighting of the likelihood of change per residue can be varied by specifying the percentage of each nucleotide to be incorporated at a given location.
For either loop diversity or extension diversity many different strategies for retaining proximity in sequence space to a favored sequence are known in the art. By way of example and without limitation, to diversify around the codon for methionine, ATG, and retain hydrophobic character, the template could be specified to contain at the first position 40% A and 20% of each other residue (C or G or T), at the second position 70% T and 10% of each other residue, and at the third position 75% of G and 25% C. Such a mixture would weight the substituted residues toward hydrophobic amino acids (which often contain a T in the second position), consistent with the hydrophobic character of methionine. The most likely encoded amino acid would be methionine itself. Depending on practical limitations for the preparation of the template nucleic acids, it may be more or less convenient to randomize each position in a manner specific for that residue, and in practice randomization schemes are frequently chosen that introduce an invariant probability of retaining one dominant residue and a fixed proportion of alternate residues, for example 70% A, 30% B for A, 70% C, 30% D for C, 70% G, 30% H for G and 70% T, 30% V for T. B, D, H and V are respectively the IUPAC codes for “not A”=C, G, or T, “not C”=A, G, or T, “not G”=A, C or T, and “not T (and not U)”=A, C or G.
The engineered transglutaminase barrel proteins of the present invention are useful for the creation of binding proteins that adsorb with high affinity to selected targets. The potential uses of such binding proteins are broad, and include, without limitation, the analytical detection and measurement of molecules or complexes of molecules, the interruption or neutralization of cellular or humoral signaling events via the blockade of enzymes, receptors or ligands, the normalization of homeostatic balance by return of functionality compromised by age, disease, or trauma, the induction of receptor-mediated signaling by receptor agonism, the targeting of undesired cells for destruction, and the localization of binding targets, for example of tumors, microbes, thromboses, or sites of tissue damage. Targets of artificial binding proteins are often proteins or peptides but can also be carbohydrates, lipids, nucleic acids, small molecules such as drugs, metabolites, or toxins, or compositions formed from small molecules or inorganic species such as either natural or synthetic polymers, glasses, metals or alloys, semiconductors or insulators; targets can also comprise modifications or substitutions of proteins, carbohydrates, lipids, or nucleic acids, or combinations of one or more such agents, such as a carbohydrate-substituted proteins, carbohydrate-substituted lipid, RNA-protein complexes, etc. Methods for identifying binding proteins that display high-affinity and high specificity for their targets are known in the art.
ETBPs of the present invention can also be used to create novel activities, such as catalytic activity, or substrate activity, based on the incorporation of prosthetic groups or designed or randomly selected sequences that can be installed in the ETBP. For example ETBPs can be used to generate substrates or inhibitors of proteases, or to provide proximity enrichment for substrates for enzymatic activities. In keeping with their utility as antibody equivalents, genetic selections or designed modifications that introduce catalytic potential can be incorporated into ETBPs through methods well known in the art, such as selection for affinity to a transition state analog of an intermediate that is found in a reaction to be facilitated by an ETBP.
To achieve high affinity and selectivity, ETBPs can be endowed with a novel surface complementary to a target of interest. For this purpose random libraries of proteins can be created and screened for rare variants that have desired properties; alternatively, specific variants can be designed by computational analysis of the target binding surface and construction of a series of candidate binding proteins that may have the appropriate behavior. Random substitution schemes can be employed when the detailed molecular structure of the target is not known, or when the most appropriate site on a structurally well-characterized molecule cannot be determined in advance. Most contemporary scaffold diversification strategies are based on random substitution. The invention further provides for mixed computational and random strategies, for example in which random diversification leads to candidates that are further optimized by directed substitution, or the use of computational techniques to predict families of candidates that can be screened for an activity of interest.
Construction of Libraries and Designed Variants
Libraries of ETBPs can be prepared in various ways known to those skilled in the art. Disseminated random substitution, clustered substitution, and designed (targeted) alteration are strategies that have been employed to increase the affinity of a given diversified scaffold for a particular target protein. In general, the objective of such diversification is to increase affinity without compromising the overall stability or solubility of the protein. One of the most widely employed strategies is surface randomization, the replacement of endogenous sequences on one particular aspect or face of a protein in order to generate a highly diverse collection of surfaces. Two common subtypes of surface randomization are loop and pocket diversification, used for proteins that are naturally convex or concave respectively. Randomizations may conserve or alter length if the scaffold is appropriately stable. In addition the natural geometry of the scaffold may be altered by incorporation of structural elements that endow the randomized or grafted sequences with particular folds or shapes. Among the known elements that may be employed for such purposes are the placement of cysteine residues such that a disulfide-linked loop is formed, the introduction of helix or sheet-destabilizing residues, such as glycine or proline, the incorporation of beta turns or Trp cage motifs, or the formation of additional secondary structure elements, such as short alpha helical or beta strand sequences.
The affinity and stability of loop-substitution surface randomized ETBPs can be further improved by the inclusion of mutations in the beta strands that improve rigidity or alter the positioning of the loops. Such favorable non-contact site mutations are well-known in the art and can be discovered by random mutagenesis once an initial candidate has been identified. Typically mutagenesis of the entire ETBP is performed, with selection for variants that exhibit higher binding affinity.
The engineered transglutaminase barrel proteins of the present invention can be further adapted to include diverse polypeptides sequences at their amino or carboxyl termini. The additional diversity may enhance affinity by providing secondary binding sites to the target, or may enhance the functional properties of the protein by binding to proteins with enhanced plasma half-life, or proteins that are known to be enriched in the vicinity of the target, or that afford the possibility of concentration in an organ or tissue-specific manner by binding to organ or tissue-specific secondary targets. When additional diversity elements are incorporated at the amino or carboxyl termini, measures may have to be employed to protect those elements from naturally occurring exopeptidases, such as the peptidyl peptidases, aminopeptidases, carboxypeptidases, and related enzymes. Methods of predicting and defeating susceptibility to exopeptidases are well known in the art. Methods of blocking exopeptidase activity include amino and carboxyl-terminal modification, incorporation of additional residues that are not substrates for the exopeptidases, or chemical modifications that destroy susceptibility.
The identification of a high-affinity, high selectivity ETBP can be achieved by either screening methods or selection methods. A screening method typically requires two elements: a supply of candidate ETBPs to be tested for affinity to the target; and a systematic method for the enumeration of the candidates, such as an ordered array or systematically composed mixture that can be deconvolved to reveal the identity of the most active variants. Screening methods often require that large numbers of ETBPs be evaluated; in such cases it is common to use pooling schemes to mix candidates, allowing the presence or absence of a desired candidate to be determined with fewer measurements. Active pools are further subdivided to identify active unique species. Candidates derived from such screens can be subjected to further randomization and screening to progressively derive ETBPs of higher binding affinity.
Selection methods typically require a library of candidate ETBPs, each prepared in a form that provides a genetic linkage between the protein and a nucleic acid that encodes or identifies the protein. A mechanism must be provided to physically isolate and purify candidate binding proteins and their associated nucleic acids from the remaining library members that lack activity. In selection methods many fewer measurements are typically performed than in screening methods.
The present invention further provides methods for the identification of ETBPs having favorable affinity, selectivity, solubility, and thermal stability. Numerous selection methods for the enrichment of nucleic acids encoding proteins of interest that bind to a specific target are known in the art and are useful for the generation of the desired ETBPs. Among these are the so-called display technologies, including phage display, yeast display, bacterial display, viral display, mammalian cell display, ribosome display, RNA display and DNA display. For the application of a particular form of display, an appropriate vector must be provided that is suitable for the display of the ETBP in the context in which selection is to take place. For example for commonly practiced forms of bacteriophage display, a plasmid encoding a translational fusion between a solvent-exposed phage structural protein and the ETBP must be created. For cellular display, such as bacterial, yeast or mammalian cell display, a fusion or stable association is created between a surface protein and the ETBP. For ribosome or mRNA display, a fusion or stable association must be created between the diversified binding protein and the mRNA that encodes it. For DNA display a fusion or stable association must be created between the ETBP and a high affinity, typically site-selective, DNA-binding protein. For some types of selection method, physical association of the binding protein and the nucleic acid that encodes it is provided by physical compartmentalization. For example, in emulsion selection methods, a small aqueous droplet is provided in which the ETBP is synthesized from a template nucleic acid. In this case the physical association is provided by the compartmentalization afforded by the nonaqueous phase that separates the individual droplets.
Display-based selections consist of one or more cycles of enrichment, each of which comprises: (i) contacting the target of interest with a mixture of diversified proteins in display context, e.g. as phage particles, cells, or RNA fusions; (ii) physically separating those phage particles, cells or RNA fusions that bind the target from those that do not bind the target, or bind less avidly, and (iii) amplifying the resulting isolated binding population by in vivo or in vitro methods to generate a new, enriched collection of diversified proteins that can be subjected to additional rounds of contact and purification. For display-based selections it is a requirement that the target permit physical isolation of the complex of target and ETBP. For example the target may be labeled with an antibody domain, peptide tag, fluorophore, biotin, or other affinity or labeling moiety, allowing the complex of ETBP and target to be physically separated from ETBPs that do not interact with the target. Alternatively antibodies or binding reagents specific for the target can be employed to effect separation. Often it is necessary to exclude unwanted ETBPs, for example those that bind to extraneous portions of the target, or to components of the apparatus used to effect physical separation. Common separation strategies rely upon an affinity matrix for the antibody domain, peptide tag, biotin, epitope or affinity moiety, such as a bead or magnetic particle bearing the cognate binding element for such antibody domain, tag, biotin, epitope or affinity moiety. Examples of commonly encountered binding elements include protein A, streptavidin, monoclonal or polyclonal antibodies, and coordinated transition metal divalent cations. Alternatively, separations based on fluorescence detection and sorting can be used. Such separations typically distinguish the signal conveyed by a fluorescent moiety or fluorophore attached to the target, and permit the identification and selective separation of cells or particles bearing high concentrations of the target by fluorescence-activated cell sorting. The contributions of undesired ETBPs can be reduced by preabsorption steps that mimic target exposure and enrichment, but are conducted in the absence of target.
Affinity
Selections or screens for ETBPs having the desired binding can be carried out by the methods described above followed by methods to identify candidate ETBPs of particular interest according to their affinity, activity, selectivity, solubility, or thermostability. Many methods for the measurement of affinity are known in the art and include solid phase as well as solution phase measurements of association constant or reaction on and off rates for combination of the ETBP with a target, or for the measurement of the catalytic activity of a catalytic ETBP. From the analysis of such equilibrium or kinetic constants the affinity of the ETBP for its target can be measured. Some methods of measuring affinity include, solid phase assays, such as planar or bead format assays, solution phase assays, or cell-based assays. Detection in such assays can be based on the analysis of changes in a signal generated by a detectably labeled target or ETBP, such as a radiolabeled target or ETBP or targets or ETBPs conjugated to or associated with an enzymatic activity or a fluorophore or fluorescent protein, or an active prosthetic group that behaves as a catalyst for a reaction or a change in property that is easily monitored. Common methods for measuring affinity include radiolabel or enzyme-linked immunosorbent assays, or assays based on surface plasmon resonance, fluorescence resonance, fluorescence polarization, or fluorescence autocorrelation spectroscopy or microscopy. A common form of affinity measurement is one in which target is immobilized on the solid phase, and varying concentrations of a solution containing a detectable form of the ETBP is contacted with the immobilized target to measure the amount of ETBP bound as a function of ETBP concentration.
Activity
For therapeutic purposes it is often useful to determine the activity of a particular ETBP for its proposed utility. For example, if the ETBP is to exert a therapeutic action by inhibiting the binding of an enzyme to its substrate or a ligand to its receptor, candidate ETBPs with affinity for the enzyme, ligand or receptor can be tested for their ability to inhibit the functional association that should be compromised for the desired effect. A bioassay is often used to determine the activity of a candidate ETBP, in which a cellular process or an in vivo response is measured in the presence or absence of progressively greater amounts of the ETBP of interest.
Selectivity
ETBPs according to the present invention may bind to single members of families of targets, or multiple members of families of targets, to achieve the desired therapeutic, analytical, manufacturing, or research utility. For example, the neutralization of biological activity for therapeutic purposes may optimally require the antagonism of more than one target, or the quantitation of such biological activity for analytical purposes may require the recognition of more than one target, or the purification of some targets of interest may require the recognition of families of related molecules. The selectivity of candidate ETBPs can be manipulated during selection or screening by including comparator targets for which binding affinity is either desired or not desired. For example, to create a highly selective ETBP that recognizes one member of a multimember family of targets, such as family of closely related proteins, a preselection can be made with the undesired targets, discarding the so-selected ETBPs, followed by a selection with the desired target. Or the activity of the ETBP identified by selection or screening methods can be assessed by comparing the binding affinity to the desired target with that of unrelated targets or related targets for which affinity is either desired or not desired. Such screening methods need not provide precise information, but for convenience may convey simple approximate measures of relative affinity, for example based on signal strength in an assay format similar to that of an enzyme linked immunosorbent assay (ELISA).
Solubility and Stability
Candidate ETBPs of the present invention that have been identified by selection or screening can be further evaluated and modified if necessary for additional properties that are required for the field of use. For example, for the manufacturing of ETBPs intended for most uses, a candidate ETBP can be highly soluble and thermostable. Methods are provided by the present invention for the evaluation of the solubility and thermostability of ETBPs as well as their suitability for expression in properly folded form in E. coli. In general methods for the evaluation of thermostability are well known in the art, and consist of thermal stress testing or extended storage testing at defined temperatures, followed by measurement of binding activity. In some cases a test for relative thermostability can be as simple as the measurement of the fraction of ETBP remaining soluble following incubation of the ETBP for a defined time at a particular temperature. Another suitable method for measuring thermostability is differential scanning calorimetry. Methods for the indirect assessment of folded status of proteins in E. coli are also known in the art, and in the present invention comprise fusion of the candidate ETBP to an easily monitored protein whose activity is only apparent in its properly folded form, such as GFP or an antibiotic resistance. The relative degree of folding has been found by others to be a property shared by both domains of a fusion protein in E. coli, so that if the ETBP moiety is not properly folded, the likelihood that the GFP or antibiotic resistance moiety will be folded is commensurately low. In such cases cells expressing inactive or improperly folded ETBP fusion proteins will not show high green fluorescence or high antibiotic resistance.
Compositions
Compositions of nucleic acids and polypeptides, as well as substituted nucleic acids and substituted polypeptides, are included in the present invention.
Substituted ETBPs
Substituted ETBPs according to the present invention can be created by site-specific targeting methods that are either chemical or enzymatic in nature. Substitution may be provided either in vivo or in vitro, and can endow the nucleic acid or polypeptide with additional features useful for selection, purification, or therapeutic, analytical, manufacturing or research utility. Exemplary substitutions that are frequently encountered in the art include prosthetic groups, such as biotin and lipoic acid, N-terminal modifications, such as various amides and cyclic amides, alkylations of cysteine or selenocysteine residues, replacement of natural with unnatural amino acids, as for example may be practiced by proteolytic resection and replacement, the formation of isopeptide bonds such as are formed between glutamine and lysine residues, polymer substituents, such as polyalkylene glycols of varying lengths and/or branch structures, small organic molecule substituents, including receptor or ligand binding molecules or their fragments, and various other peptide or nonpeptide adducts that may be attached to the ETBP for a particular utility. The substitutions may aid in the detection, purification, or localization of the ETBPs and may confer favorable properties by virtue of such localization. For example substituted ETBPs may bind to plasma or cell surface proteins to extend the half life of the substituted barrel, or may be concentrated on the surface of particular cells, or in certain organs or tissues, by virtue of their affinity for cell-, organ- or tissue-selective secondary targets, such as receptors, carbohydrates, lipids or combinations of such secondary targets as may be presented by the organism to be treated or exposed to the substituted barrel.
The engineered transglutaminase barrel proteins of the present invention may be delivered as pure proteins or may be generated in situ, for example following delivery by a gene targeting or genetic therapy that may involve the in vitro or in vivo modification of the genetic complement of an existing cell to produce the polypeptides of the present invention.
Substitution or Modification to Achieve Extended In Vivo Half Life
Engineered transglutaminase barrel proteins may be modified to extend their plasma half life, for example by modification with polyethylene glycols, by translational fusion or post-translational crosslinking to naturally occurring plasma proteins having extended half life or by a combination of such methods. Suitable naturally occurring plasma proteins having extended half life include antibodies, albumin, apolipoproteins, serpins and some constituents of the complement and coagulation cascades. For example, translational fusion to human antibody Fc domains, particularly human IgG1 Fc domain, has been a widely used to improve plasma persistence of proteins. ETBPs may also be endowed with extended half life by dimerization or multimerization to produce polypeptides that are too large to be naturally subject to renal filtration. Suitable methods for dimerization or multimerization include disulfide bond formation, translational fusion, and chemical or enzymatic crosslinking that is either site-selective or site-nonselective. When translational fusion is employed it may be necessary to provide one or more flexible linkers connecting monomers of the diversified barrel. Such flexible linkers are well known in the art and may consist of a plurality of glycine residues in combination with chiral amino acids that provide favorable aqueous solubility, such as the charged or uncharged hydrophilic amino acids aspartic acid, glutamic acid, arginine, histidine lysine, serine, threonine, tyrosine, asparagine, or glutamine. Flexible linkers need not be confined to glycine and hydrophilic residues so long as the linker so constructed does not confer otherwise unfavorable biophysical properties on the ETBP, such as poor solubility, instability to aggregation, or susceptibility to proteolysis.
The formation of antibodies against ETBPs of the present invention can be minimized by the attachment of polymers, such as polyethylene glycols of varying size, branching morphology and attachment groups, to the barrels, their N terminal or C terminal extensions, or their prosthetic groups, using either site-selective or site-nonselective methods.
Additional Uses of ETBPs
Therapeutic Uses
The engineered transglutaminase barrel proteins of the present invention can be used as targeting principles to deliver other therapeutic or analytical elements to an organism in need of therapy or diagnosis. For example, they may be attached to highly active cytostatic or cytotoxic agents to effect the growth arrest or elimination of an undesired cell type, such as a neoplastic or pre-neoplastic cell, or for the reduction in mass of a hypertrophic tissue or organ such as a hypertrophic prostate, or for the elimination of populations of immune cells that are undesired, for example those promoting or causing autoimmune syndromes. Such cytostatic or cytotoxic agents may be synthetic or natural small molecules, for example, maytansine and its derivatives, anthraquinones, alkylating agents such as cyclophosphamide or its prodrug forms, tubulin-binding agents, geldanamycin or its derivatives, or enediyne antibiotics such as calicheamycin, among others. The cytostatic or cytotoxic agents may also be proteinaceous toxins or combinations of small molecules and proteinaceous toxins. Proteinaceous toxins that may be employed for the purposes of cytostatic or cytotoxic therapies include bacterial toxins, members of which can be drawn from the 339 or more species or families that have been identified to date, including their natural variants, serotypes, isoforms, and allelic forms from both Gram-positive and Gram-negative bacteria (Alouf and Popoff, Eds. “The Comprehensive Sourcebook of Bacterial Protein Toxins, 3rd Ed.” Academic Press. 2006). Exemplary bacterial toxins widely used for selective ablation of target cells or tissues include diphtheria toxin, Pseudomonas exotoxin A, anthrax lethal factor, and aerolysin.
Bispecific ETBPs
Dimeric or higher multimeric ETBPs can be used to juxtapose cells or induce cellular actions by receptor crosslinking that may have a favorable therapeutic effect. For example, therapeutic strategies aimed at amplifying the cytotoxic action of macrophages, natural killer cells, or cytotoxic T cells have been described which rely upon the use of bispecific antibodies or related compositions. Such bispecific antibodies typically provide one antibody combining site that recognizes a target on the cell type to be ablated, and a second antibody combining site that recognizes a cell surface receptor on macrophages, natural killer cells, or T cells that, if engaged, induces the cytolytic effector program of those cells, leading to destruction of the target. Alternate forms of bispecific antibodies promote the selective disabling of responses by mast cells or B cells by producing crosslinks between activating receptors, such as the high affinity receptor for IgE on mast cells or the immunoglobulin receptor on B cells, and inactivating receptors, such as the inhibitory receptor for immunoglobulin FcγRIIB, also found on mast cells and B cells. The coordination of the activating receptor and the inhibitory receptor frustrates the signals emanating from the activating receptor, resulting in a favorable therapeutic effect. Similar bispecific compositions can be provided by the ETBPs of the present invention, which can be joined by a variety of methods to provide bi- or multi-specific binding principles for therapeutic treatments.
In addition ETBPs of the invention may be used to target prodrug activating enzymes to cells, tissues or organs that are desired to be made subject to the action of the drug of interest. Such uses of ETBPs follow closely the uses of antibodies in antibody directed enzyme prodrug therapies, which rely upon antibodies for the targeting of a prodrug metabolizing moiety which converts a circulating prodrug to an active form. Such local activation schemes have been developed to allow a more specific delivery of highly toxic chemotherapeutic agents to the vicinity of a neoplastic cell in vivo.
Engineered transglutaminase barrel proteins can also be used to deliver bioactive principles to a cell, organ, or tissue that is desired to be targeted. To produce interference with the function of a particular pathway that may be essential systemically but undesired in a specific organ, for example to block a hepatic action but not a central nervous system or renal or muscular action, an engineered transglutaminase barrel protein can be used to convey an antagonist of that pathway to a specific tissue, by (as in the example cited) binding to a liver-specific cell surface protein. A bioactive principle delivered by the ETBP can be attached to the ETBP by translational fusion or by chemical or enzymatic modification in a site-selective or site-nonselective manner.
Diagnostic Uses
ETBPs of the present invention can be used as antibody equivalents for many assay purposes. ETBPs can serve as the capture or detection reagent for ELISA-type assays or as the detection reagent for ELISpot assays or for the enumeration of protein abundance by flow cytometric measurement technologies. ETBPs can be conjugated to fluorophores, fluorescent proteins or enzymes to aid in the detection and/or quantitation of analytes of interest. Translational fusions of ETBPs to enzymes or other proteins that aid in the detection of analytes can be made and the resulting fusions can be expressed in prokaryotic or eukaryotic cells to provide a convenient renewable source of reagent. The favorable thermostability properties of ETBPs allow their use in arrays of analyte detector, for example in the planar format of protein binding arrays, or in the bead format of multiplexed fluorophore ratio indexed bead systems, such as the Luminex system. Detection of analyte binding with an ETBP can follow many of the assay format designs and detection schemes that have been disclosed for high sensitivity and selectivity detection by antibodies, such as light scattering, light surface plasmon scattering, fluorescence polarization, time resolved fluorescence, fluorescence autocorrelation, electroluminescence, chemiluminescence, fluorescence resonant energy transfer, fluorescence quenching or unmasking, coagulation or flocculation of beads, cells or other particles, or by providing nucleic acid or modified nucleic acid tags for detection by amplification methods including polymerase chain reaction, ligation-mediated probe amplification, branched nucleic acid assay, or isothermal amplification, with or without a ligation step; or by conveying enzymatic activities detectable by absorbance, fluorescence, evanescent field or surface potential perturbation. Monospecific or multispecific ETBPs can be prepared to identify unique analytes or families of analytes. In addition, monomeric, or multimeric ETBPs can be used as capture or detection reagents.
Labeled ETBPs can be used to image diseased cells, tissues or organs, either in vivo or in vitro. ETBPs can be conjugated to radionuclides, or to prosthetic groups incorporating or binding to other molecules comprising radionuclides. Common radionuclides used in imaging include F-18, I-131, I-123, Tc-99m, In-111 or Ga-67. Alternatively ETBPs can be conjugated to groups enclosing caged hyperpolarized xenon, or can be joined or attached to beads, nanoparticles or nanocrystals susceptible to detection by magnetic resonance imaging. Radionuclides can be detected by nuclear scintigraphy using equipment and methodology well known in the art, such as gamma cameras and positron emission tomography. In addition, images obtained by one modality, such as magnetic resonance imaging can be superimposed on images obtained by other modalities, such as nuclear scintigraphy, or two or more radionuclides of different spectral properties can be combined with different ETBPs, to permit better localization of images and more precise staging or diagnosis of disease conditions. Uses of such conjugated ETBPs include the in vivo imaging of tumors, infections, regions of ischemic damage or poor perfusion, clots, bone or eroded bone, sites of inflammation or degeneration, accumulations of amyloids, paraproteins or prion proteins, or to interrogate the status of prosthetic devices and/or their interfaces with normal or diseased tissue. ETBPs labeled with enzymes, fluorophores, fluorescent proteins, ferritin, gold or silver particles, or electron dense beads, can be used in conjunction with microscopic or ultramicroscopic techniques to diagnose pathological conditions or to identify, enumerate or quantitate the burden of relevant targets that signify the disease status of the cells, tissues, organs or organisms being studied.
The imaging of tissues using labeled or conjugated ETBPs can be used to guide diagnostic or therapeutic procedures, such as biopsies, resections, radioablations, radiotherapy, or locally delivered chemotherapy.
Manufacturing Uses
The favorable thermostability and solubility properties of the ETBPs of the present invention also permit their use as adsorption reagents for the purification of proteins and complex biological structures, such as vaccine components. The positive manufacturing economies of prokaryotic production allow ETBPs to be used in settings for which the routine use of antibody reagents or materials would be considered prohibitively expensive.
Typically, for a manufacturing use an ETBP having the desired selectivity, solubility, thermostability, and affinity for a target will be prepared in a form that allows its constitution into an adsorbent, which may comprise a column medium, bead, or coated surface to which a target stream is to be exposed. Following adsorption of the target to the solid support, the nonbound material will be removed by one or more washing steps and the desired target material will be eluted, typically by raising or lowering the pH, as is common in the elution of antibody-based affinity supports. Various hydrophilic matrices that are used as supports for such affinity media are well known in the art and includes various, typically porous and crosslinked, polymers, such as crosslinked agaroses, dextrans, acrylamides, hydrophilic acrylates, or inorganic matrices such as controlled pore glass, or nonporous but fine particles such as magnetic beads, and functionalized or surface passivated silica or cellulose particles. ETBPs can be attached to such media by methods such as electrophilic attack by aldehydes, oxiranes, activated carbonates, iminocarbonates, cyanate esters, haloacetamides, maleimides, or activated esters, including carbodiimide activated carboxylic acids. Many commercial suppliers of pre-activated media suitable for attachment of the ETBP are known. In addition the ETBP can be engineered by the incorporation of specific residues or sequences that favor the attachment of the ETBP to the media in an ETBP site-selective manner. For example, the incorporation of cysteine or selenocysteine residues, or substrate sequences for transglutaminases or sortases can be used to provide specific sites at which the ETBP can be linked to a solid support.
Research Uses
Research and analytical uses of ETBPs include the replacement of antibodies for detection and quantitation of analytes in various contexts, for example in immunoblotting, ELISA, ELISpot, flow cytometry, bead-based coagulation or detection systems, for detection of analytes by light scattering, surface plasmon scattering, chemiluminescent or electroluminescent detection, by fluorescence polarization, time-resolved fluorescence, fluorescence autocorrelation, fluorescence resonant energy transfer, or fluorescence quenching or unmasking. ETBPs can be conjugated with various fluorophores or fluorescent proteins to provide probes for the presence or absence of analytes. The analytes may include proteins, carbohydrates, nucleic acids, lipids, small molecules of natural, synthetic or semisynthetic origin, as well as polymers, glasses, metals and alloys, or combinations of these. ETBPs can be conjugated to enzymes, proteins, nucleic acids, carbohydrates, lipids, polymers, small molecules of natural, synthetic or semisynthetic origin, to provide an analyte detection method or additional functionality, or can be endowed with additional substituents having utility for detection or amplification of signal, such as by providing covalent or stable noncovalent attachment of nucleic acid or modified nucleic acid tags for detection by amplification methods including polymerase chain reaction, ligation-mediated probe amplification, branched nucleic acid assay, or isothermal amplification, with or without a ligation step. ETBPs can be adsorbed on solid surfaces, such as plates, trays, capillaries, fabrics, flexible or rigid sheets, beads, or particles, all of which may provide either surfaces for noncovalent absorption or chemically activated surfaces for covalent attachment. Such ETBP-substituted surfaces may be used to provide either capture reagents, or in the case of bead or particulate adsorbed material, detection reagents. Examples of uses of labeled ETBPs include, without limitation, microscopy, ultramicroscopy, flow cytometry, flow microscopy, immunoblotting, immunoprecipitation, spectroscopy, or in vivo imaging.
Methods of Preparation
ETBPs of the present invention are often easily prepared by expression in prokaryotic cells, such as E. coli. Moreover ETBPs often have unusual and favorable solubility properties that allow them to be readily purified using simple scalable steps amenable to high volume manufacturing. ETBPs according to the present invention frequently present a pH-dependent solubility profile that is opposite to that of many E. coli proteins. At neutral pH and in moderate ionic strength, ETBPs often have moderate to poor solubility, whereas ETBPs are very soluble and retain their structures under acidic conditions, for example in 10-100 mM HCl. Accordingly ETPBs can often be purified without recourse to chromatography by the simple expedient of alternating the pH of an aqueous solution or suspension of ETBPs. A few successive alternations suffice to afford highly pure ETBP in many cases. ETBPs can be further purified by conventional methods, for example to remove endotoxin or remaining contaminant proteins, if desired. Fusion proteins bearing ETBP moieties often behave with respect to alternations of pH as the ETBPs themselves, so that for example an ETBP-GFP fusion protein can frequently be easily prepared from E. coli by harvesting an insoluble pellet of ETBP-GFP fusion protein produced by fermentation and extracting soluble protein in dilute HCl. This property of ETBPs is not confined to engineered transglutaminase barrel proteins, but is often found among unmodified barrel proteins. The ability to form fusions that can be easily purified without recourse to chromatographic methods is highly attractive for numerous applications in biotechnology, particularly those that require inexpensive high volume production of pure materials. Accordingly the present invention also provides methods for the purification of fusion proteins comprising unmodified or native transglutaminase barrel proteins.
The invention will now be further illustrated by the following non-limiting examples.
EXAMPLES
Example 1
Characterization of Wild Type F13β2 Scaffold
Alternative scaffolds for therapeutic applications are preferably prepared in high yield and purity by prokaryotic fermentation. To characterize the ability of an isolated barrel domain to autonomously fold in E. coli a fusion of the human β-barrel 2 domain joined at its C-terminus to GFP was studied. For this purpose wild type human factor XIIIA β-barrel 2 domain (positions T629-R729) was amplified from full length FXIII-A cDNA clone (SC120099; OriGene Technologies, Inc., Rockville, Md.) by PCR with primers oligo17 and oligo 18rev (all primers used for cloning are presented in Table 10). An initiator methionine codon was introduced at the location corresponding to the N-terminus of the protein and the final fragment was cloned into Nde I and Bam HI cloning sites of modified pet28(a+) vector, containing GFP protein. The map of the plasmid (modified pet28(a+)-F13β2-GFP) is shown in FIG. 3A. The correct clone was confirmed by sequencing (FIG. 4). The sequence corresponded to the known sequence of factor XIII-A (accession number NP_000120) with one change (T650I) which is consistent with existing polymorphism reported within the FXIII-A (Gerhard et al, Genome Res. 2004, 14:2121). The three residues at the C-terminus (P730-M732) were not included in the F13β2 scaffold construct because they are not resolved in the crystal structure and hence can likely adopt multiple configurations in solution. Also, for selections involving C-terminal extensions the sharp turn at the proline residue (P730) could potentially bring the extended portion into close proximity with loops of the scaffold and interfere with binding to a target. Re-introduction of the P730-S731-M732 sequence into the C-terminus of selected binder proteins can be performed after selection (see below).
The resulting isolated Factor XIII β2 barrel protein is a small domain of 102 amino acids with methionine at the first position and R102 (corresponding to R729 of FXIII-A) at the C-terminus. The wild type Factor XIII β2 barrel protein GFP fusion was expressed in E. coli BL21 (DE3) pLysS cells (Invitrogen). In brief, the cells were grown on a nitrocellulose filter placed on LB agar plate (containing 50 μg/mL kanamycin) overnight at 37° C. Protein expression was induced by transferring the nitrocellulose filter with cells onto a new plate containing 1 mM IPTG and incubation for additional 3 hours at 37° C. The level of folded protein expression was assessed visually under UV light. Alternatively, BL21 cells containing F1362-GFP construct were grown in LB culture (containing 50 μg/mL kanamycin) and induced at A600 ̃0.6 with 1 mM IPTG. After 3 hours incubation at 37° C. the cells were pelleted by centrifugation at 10000 rpm for 3 minutes at 4° C. and resuspended in 1×PBS buffer (137 mM NaCl, 2.7 mM KCl, 10 mM sodium phosphate, pH 7.4) to A600=1.5. GFP fluorescence was measured using a Molecular Devices spectrofluorimeter (Aex=490 nm; Aem=510 nm; 5 nm bandwidth). It has been shown that when various proteins are expressed in E. coli as an N-terminal fusion with GFP, folding and formation of the GFP chromophore is related to the correct folding of the upstream domain. When the isolated Factor XIII β2 barrel protein was expressed as a fusion to GFP, a high yield of green fluorescence was observed, indicating the scaffold is likely folded properly (FIG. 5A). The F1362 scaffold behaves similarly to another well characterized alternative scaffold, the wild type 10Fn3 domain (Koide et al, J. Mol. Biol. 1998, 284:1141), whereas a control mutant Fn3 with disrupted hydrophobic core was not folded.
The solubility of the expressed protein was also assessed by SDS-PAGE analysis of total and soluble fractions. The cell pellet from 1 ml culture was collected, and after freeze/thaw disruption, resuspended in 1×PBS (pH 7.4), containing LysonaseTM Bioprocessing Reagent (EMD Chemicals Inc., San Diego, Calif.). After incubation at room temperature for 20 minutes CHAPS was added to 0.4% final concentration. The solution was rotated for 20 minutes at 4° C., and the soluble fraction was separated by centrifugation. Aliquots of total and soluble fractions were collected before and after centrifugation for SDS-PAGE analysis. Additionally the fluorescence was monitored under UV light in cell pellet, insoluble pellet and soluble fraction. Characterization by SDS-PAGE showed that the F13β2-GFP fusion is soluble at 37° C. (FIG. 5B).
Optimal protein expression is required for in vitro protein production during selection and for E. coli expression in development stages of therapeutic product. Therefore, the F13β2 sequence was optimized for both mammalian and E. coli expression prior to further scaffold engineering. Changes were made based on Codon Usage Database developed by Yasukazu Nakamura (www.kazusa.or/jp/codon/readme_codon.html). During the first step of construction three fragments (ABC, CDE and EFG) were obtained by PCR using overlapping primers: oligo 19 and oligo 20rev; oligo 21 and oligo 22rev; oligo 23 and oligo 25rev, respectively. The fragments were mixed in equimolar ratio and PCR was performed with flanking primers oligo 53 and oligo 28rev to introduce NdeI at 5′ end, and His6-tag-Stop sequence and Barn HI site at 3′ end. The final fragment was cloned into modified pet28(a+) vector. The map of the plasmid (modified pet28(a+)-F13β2-His6 (XhoI) (sequence set forth in Table 14)) is shown in FIG. 3B. Codons for 20 amino acids were changed and the new sequence was used for further constructions (FIG. 4). The optimized single F13β2 domain, containing His6 sequence on the C-terminus was expressed for 3 hours at 37° C. or overnight at 18° C. in E. coli as described for F13β2-GFP fusion, and total and soluble fractions were analyzed by SDS-PAGE. For His-tag affinity purification the supernatant was rotated for 1 hr at 4° C. with Ni-NTA agarose (Qiagen) preequilibrated with buffer C (1×PBS [pH7.4], 0.5 M NaCl; 20 mM Imidazole, 0.3% CHAPS). The resin was washed with 50 column volumes of buffer C and 30 column volumes of buffer D (1×PBS [pH7.4], 20 mM Imidazole). The protein was eluted with 1×PBS, 200 mM Imidazole (pH 7.4) and was dialyzed against 1×PBS at 4° C. The protein concentration was measured using Coomassie (Bradford) protein assay reagent (Pierce Biotechnology, Inc., Rockford, Ill.). The F13β2 scaffold was soluble (FIG. 5B) when expressed at 37° C. and 18° C. yielding approximately 5-10 mg of pure protein from one liter culture by affinity chromatography via a His6 tag.
Example 2
Single Cysteine Mutagenesis of F13β2 Domain
The F13β2 scaffold has a single cysteine at position 69 with a side chain directed towards the hydrophobic core of the domain (FIG. 6A). Because free cysteines can sometimes pose challenges in selection, expression, or purification, we performed NNS randomization of C69. A two-step PCR was performed using the codon-optimized F13β2 clone as a template. During the first step, two fragments were obtained using two pairs of primers: oligo 17/oligo 22rev and oligo 51/oligo 52rev. The fragments were recombined in a second PCR with flanking primers oligo 17 and oligo 52rev and the final fragment was cloned into NdeI and BamHI sites of a modified pet28(a+) vector containing GFP (Waldo et al, 1999). F13β2 C69X mutants were expressed as N-terminal GFP-fusions in E. coli and the correct folding and solubility of the proteins at 37° C. in 1×PBS were assessed by GFP fluorescence of colonies growing on agar and by SDS-PAGE of total and soluble fractions as described above for wild type F13β2 scaffold (FIG. 6B). Substitutions with large or hydrophilic amino acids (tryptophan, asparagine) or the constrained amino acid proline resulted in a loss of solubility following expression at 37° C. A C69T mutant was completely soluble whereas a C69S substitution was only partially soluble at 37° C. The aliphatic hydrophobic amino acids valine, leucine and alanine were found to be useful to replace Cys69 without changing solubility. A single F13β2C69V-His6 (“His6 disclosed as SEQ ID NO: 29) domain was generated by PCR using primers oligo 53 and oligo 28rev (Barn HI), and was shown to be soluble when expressed at 37° C. and 18° C. (FIG. 6B). Subsequently, the Factor XIII β2 barrel protein bearing a C69V mutation was employed in F13β2 library construction.
Example 3
Point Mutagenesis of F13β2 Scaffold at W38 and Y92
Randomization of an alternative scaffold can be performed by diversification of the surface of a known ligand or receptor binding site or by diversification of surfaces not known to participate in ligand or receptor binding. Preferable randomization strategies minimize changes that destabilize the scaffold core. The data obtained with Cys69 substitutions illustrate that core substitutions of transglutaminase barrel proteins may have reduced solubility and/or stability if they result in energetically unfavorable structures. To test the utility of randomization of scaffold sequences not contributing to the integrity of the beta barrel, we randomized two hydrophobic residues in F13β2 domain that are predicted to have solvent-exposed side chains: W38 and Y92. These residues are positioned in close proximity to the rest of FXIII-A protein and could possibly be involved in hydrophobic interactions with other domains of FXIII. Changing bulky solvent-exposed hydrophobic residues could potentially produce a more stable, soluble scaffold. The codon-optimized F13β2 clone was used as a template for two-step PCR. Initially, two fragments were prepared for W38X randomization using two pair of primers (oligo 17/oligo 48rev and oligo 49/oligo 52rev). The fragments were recombined in second PCR with flanking primers oligo 17 and oligo 52 rev. To introduce Y92X randomization PCR product was produced with oligo 17 and oligo 50rev and extended with flanking primers oligo 17 and oligo 52 rev. The final fragments were cloned into NdeI and BamHI sites of modified pet28(a+) vector. The F13β2 mutants were expressed as N-terminal GFP-fusions in E. coli BL21 and the correct folding and solubility of the proteins at 37° C. in 1×PBS were assessed by GFP fluorescence of colonies growing on agar and by SDS-PAGE of total and soluble fractions as described above for wild type F13β2 scaffold. Most of the W38X (9) and Y92X (12) mutants were soluble, resembling the wild type F13β2 domain (Table 1). Only substitution with the structurally constrained amino acid proline resulted in a complete loss of solubility, possibly due to energetically unfavorable changes in the β-sheet in which W38 is positioned. It is possible that introduction of a sharp turn into β-strand via proline could disrupt the integrity of the domain. All other changes were well-tolerated by the scaffold which was a good indication for further randomization studies.







TABLE 1







Solubility profile of F13β2 mutants.











Mutation (X)
W38X
Y92X







Ala
yes (partially)
yes



Arg
N.D.
yes



Asn
N.D.
yes



Asp
N.D.
yes



Cys
yes (partially)
N.D.



Gln
N.D.
N.D.



Glu
N.D.
N.D.



Gly
yes (partially)
yes



His
yes
yes



Ile
N.D.
yes



Leu
yes
yes



Lys
N.D.
yes



Met
N.D.
yes



Phe
yes (partially)
N.D.



Pro
no
N.D.



Ser
yes (partially)
yes



Thr
N.D.
yes



Trp
yes
N.D.



Tyr
N.D.
yes



Val
yes
N.D.







F13β2-W38X and F13β2-Y92X mutant proteins were expressed as GFP fusions in E. coli at 37° C. and their solubility in 1xPBS, pH 7.4 was assessed by GFP fluorescence and SDS-PAGE (N.D.—not determined).






Example 4
Loop Diversification of the F13β2 Scaffold
Several loop diversification schemes were designed based on the crystal structure of the A subunit of factor XIII. Loop diversification can be carried out by complete randomization or by retention of specific residues that might play a structural role in the stability of the loop or protein fold, such as loop residues for which the side chains extend toward the core. Six potential randomization loops are present in F13β2 structure: three (BC, DE, and FG) at the N-terminus and three (AB, CD, and EF) at the C-terminus of the domain (FIG. 7 and FIG. 8).
All F13β2 libraries were based on the sequence of optimized F13β2C69V clone and constructed by two-step PCR using synthetic randomized oligonucleotides. For N-terminal loop libraries three loop regions—BC, DE, and FG, were randomized by using NNS (C-NNS BC loop, C-NNS, C+2-NNS, A-NNS and Aext-NNS libraries) or 50% NNS, containing 50% NNS and 50% wild type F13β2 nucleotide, which resulted in 55 to 71% of random amino acid changes (A-50% NNS and A-50% FGext libraries). For C-terminal loop libraries (Ct C-NNS, Ct C+3-NNS, Ct A-NNS libraries) three loop regions—AB, CD, and EF, were randomized using an NNS scheme. During the first step of construction three fragments (ABC, CDE and EFG) were obtained by PCR using overlapping primers (200 pmol/each): C-NNS library (oligo 19 and oligo 35rev (BC C-NNS); oligo 21 and oligo 41 rev (DE C-NNS); oligo 63 (C69V) and oligo 45rev (FG C-NNS)); C+2-NNS library (oligo 19 and oligo 35rev (BC C-NNS); oligo 21 and oligo 41rev (DE C-NNS); oligo 63 (C69V) and oligo 46rev (FG+2 C-NNS)); A-NNS library (oligo 19 and oligo 34rev (BC A-NNS); oligo 39 and oligo 40rev (DE A-NNS); oligo 63 (C69V) and oligo 44rev (FG A-NNS)); A-50% NNS library (oligo 19 and oligo 55revl (BC A-50% NNS); oligo 39 and oligo 56revl (DE A-50% NNS); oligo 63 (C69V) and oligo 57revl (FG A-50% NNS)); Aext NNS library (oligo 19 (671) and oligo 68revl (BC Aext-NNS); oligo 691 and oligo 70revl (DE Aext-NNS); oligo 63 (711) (C69V) and oligo 72revl (FG Aext-NNS)); A-50% FGext library (oligo 19 (671) and oligo 55revl (BC A-50% NNS); oligo 39 and oligo 56revl (DE A-50% NNS); oligo 63 (711) (C69V) and oligo 72revl (FG Aext-NNS)); Ct C-NNS library (oligo 33 (AB C-NNS) and oligo 20rev; oligo 37 (CD C-NNS) and oligo 22rev; oligo 43 (C69) (EF C-NNS) and oligo 25rev); Ct C+3-NNS library (oligo 33 (AB C-NNS) and oligo 20rev; oligo 38 (CD C+3-NNS) and oligo 22rev; oligo 43 (C69) (EF C-NNS) and oligo 25rev); Ct A-NNS library (oligo 32 (791) (AB A-NNS) and oligo 20rev (80revl); oligo 36 (811) (CD A-NNS) and oligo 22rev (82revl); oligo 831 (C69) (EF A-NNS) and oligo 84revl (G4S (SEQ ID NO: 32))). For C-NNS BC loop test library ABC (randomized BC loop) fragment was obtained using overlapping primers oligo 19 and oligo 35rev, and CDEFG fragment was amplified with primers oligo 21 and oligo 52rev using F13β2C69V clone as a template. For solubility characterization the loop fragments of each library were mixed in equal molar ratio and PCR was performed with flanking primers oligo 53 and oligo 52rev to introduce NdeI at 5′ end, and Barn HI site at 3′ end. F13β2 library proteins were expressed as N-terminal GFP fusions by using a modified pet28(a+) vector, in E. coli BL21 (DE3) pLysS cells (Invitrogen). The correct folding and solubility of the proteins at 37° C. in 1×PBS were assessed by GFP fluorescence of colonies growing on agar and by SDS-PAGE of total and soluble fractions as described above for wild type F13β2 scaffold. For in vitro library production the loop fragments were mixed in equal molar ratio (40 pmol/each) and after extension PCR the amplification was performed with 10-fold excess of flanking primers oligo 55 and oligo 26rev. T7TMV transcription sequence was introduced at the 5′ end and PEG6-linker annealing sequence (GCATCCGCTATTTAA) (SEQ ID NO:1) and polyA were introduced at the 3′ end of the libraries. Additionally, Flag-tag sequence (DYKDDDDK) (SEQ ID NO:2) was introduced at the C-terminus for affinity purification of mRNA fusion molecules and proteins via M2 agarose. Oligo 661, containing unique linker sequence in 5′ non-translated region, was used for amplification of Aext-NNS library, and extended 3′ end oligo 73revl for Aext-NNS and A-50% FGext libraries. G4S sequence (SEQ ID NO: 32) was introduced at the C-terminus of Ct A-NNS library with oligo 85revl. DNA sequencing of approximately 100 library members confirmed the intended mutation rate. C-NNS, A-NNS, A-50% NNS and Aext-NNS libraries contained 2.41×1013 to 9.64×1013 members. C-NNS, A-NNS, A-50% NNS and Aext-NNS libraries were used in the myostatin selections, Aext-NNS library in the selection against human FcεRI and A-50% NNS library in the selection against human β-NGF.
C-NNS BC Loop F13β2 Library.
We investigated the effect of a full loop randomization on solubility of F13β2 scaffold by introducing changes (NNS) in five residues of BC loop (P29 to T33). Clones that had not accumulated frameshift or termination mutations (“expressible clones”) accounted for 70% of the population (Table 2). They were expressed at the N-terminus of GFP in E. coli and their folding and solubility in PBS was measured by green fluorescence of folded GFP. Almost 60% of expressed proteins were soluble at 37° C. which indicated that BC loop can tolerate substantial diversity without perturbation of the F13β2 fold and that mutations can be incorporated into the loops of F13β2 domain.
N-Terminal Loop F13 β2 Libraries: C-NNS, C+2 NNS, A-NNS, A-50%-NNS, Aext-NNS, A-50% FG Ext.
N-terminal loop libraries were constructed by randomizing three loop regions (BC, DE and FG) positioned at the N-terminus of the F13β2 scaffold. Different randomization schemes were applied for construction of F13β2 libraries for selections (FIG. 7 and FIG. 8).
The conservative C-NNS library contained only 11 residues randomized with NNS: five in BC loop (P29-T33); four in DE loop (R58-S61) and two in FG loop (D86-S87). Only flexible loop regions were changed in this library to allow the minimal disturbance of the scaffold. Indeed, 46% of the expressible clones were correctly folded (Table 2). To extend the potential binding surface in this limited library insertion of two randomized residues was applied to the FG loop in C+2-NNS library, which increased the randomization surface to 13 amino acids.
Aggressive randomization was used in the A-NNS library which contained 22 amino acids randomized with NNS: nine in BC loop (T27-T33, R35-N36); seven in DE loop (R55-E56, R58-T62) and five in FG loop (S84-R89). In this library not only loop regions, but also the loop flanking residues that are facing towards the aqueous solution were randomized in order to increase the interaction space around each loop. L34 and 157 were not changed because they are facing towards the core of the domain in crystal structure of FXIII-A. The same randomization scheme was applied in the A-50% NNS library but instead of NNS, 50% of wild type F13β2 nucleotide was introduced simultaneously with 50% of N or S at each position, which resulted in 60 to 70% of random amino acid changes in variable loops.
The solubility of the library population decreased with increased randomization, but even in the aggressive A-NNS library 17% of the expressible clones were correctly folded, which would provide enough diversity for selection (Table 2).
To further increase the binding surface of the scaffold two more libraries with loop extensions were constructed. Aext-NNS library contained 24 residues randomized with NNS: seven in BC loop (L30-T33 and 3 aminoacid extension); five in DE loop (R55-E56, R58-N61) and twelve in FG loop (S85-H90 and 6 aminoacid extension). In A-50% FGext library 28 residues were randomized with BC and DE loops changed similar to A-50% NNS library and FG loop as in Aext-NNS library. Loop extensions are predicted to provide more flexible binding surfaces compared to randomization of existing scaffold structures.
C-Terminal Loop F13β2 Libraries: Ct C-NNS, Ct C+3-NNS, Ct A-NNS Libraries.
C-terminal loop libraries were constructed by randomizing three loop regions (AB, CD and EF) on the other side of F13β2 domain. Modification of the C-terminal loops would position the potential binding site at the end of FXIII-A molecule in close proximity to the C-terminus, which is the site for attachment to the nucleic acid in RNA display. To improve the consistency of behavior between fusion proteins and free proteins, additional flexible linkers may be added to distance the RNA fusion site from the binding site. Alternatively, N-terminal fusion display strategies could be useful for these libraries.
Randomization using the NNS scheme was employed in the construction of three C-terminal loop libraries. Conservative Ct C-NNS and Ct C+3-NNS libraries contained 13 and 16 randomized residues, respectively: six in AB loop (G12-T13, V15-V16, S18-D19); two in CD loop (P44-G45 and 3 aminoacid extension in Ct C+3-NNS library); five in FG loop (R70, W72-S74, H76). Aggressive Ct A-NNS library had 22 mutated residues: eight in AB loop (G12-D19); seven in CD loop (G43-P49); seven in EF loop (R70-H76).
Non-specific binding of fusion libraries to the beads or different target proteins didn't exceed 0.6% and fusion production provided sufficient diversity for further selections (Table 2).







TABLE 2







Characterization of F13β2 libraries.










N-terminal loop libraries
C-terminal loop
















C-NNS





A50% F
libraries


















BC

C + 2-
A-50%

Aext-
Gext-
Ct C-
Ct C + 3-
Ct A-


F13β2 library
loop
C-NNS
NNS
NNS
A-NNS
NNS
NNS
NNS
NNS
NNS




















Randomization
5
11
13
22
22
24
28
13
16
22


(aminoacids)


Clones tested (n)
43
67
55
66
68
76
37
n.d.
n.d.
32


Frameshifts (%)
12
34
44
30
16
68
43
n.d.
n.d.
56


Stops (%)
19
27
56
24
49
18
24
n.d.
n.d.
25


Expressible
70
39
29
45
35
13
32
n.d.
n.d.
19


clones (%)


Soluble at 37° C. in
57
46
31
30
17
n.d.
n.d.
n.d.
n.d.
n.d.


1xPBS (% of


expressible)


Insoluble at 37° C.
43
54
69
70
83
n.d.
n.d.
n.d.
n.d.
n.d.


in 1xPBS (% of


expressible)


% oligo dT (yield)
n.d.
4.8
—
2.4
3.7
1.7
1.8
n.d.
n.d.
1.5


% flag (yield)
n.d.
11.8
n.d.
12.4
9.0
6.0
9.3
n.d.
n.d.
9.0


Pmol/mL lyzate
n.d.
3.4
n.d.
2.3
1.8
0.6
1.2
n.d.
n.d.
0.8


Binding to beads
n.d.
0.30
n.d.
0.22
0.29
0.20
0.32
n.d.
n.d.
0.07


in R0 (%)


Number of targets
n.d.
4
n.d.
6
5
4
1
n.d.
n.d.
3


tested at 100-250 nM


Binding to targets
n.d.
0.33
n.d.
0.35
0.48
0.26
0.57
n.d.
n.d.
0.33


in R0 (%)









Example 5
Selection of ETBPs Against Human Myostatin
Myostatin, a member of the tumor growth factor-β family, is a potent inhibitor of skeletal muscle growth. Disruption of the myostatin gene in mice and natural mutations identified in cattle and, recently, in humans indicate that the absence of functional myostatin results in a significant increase in muscle mass. Inhibitors of the myostatin signaling pathway are being investigated for the treatment of muscle wasting diseases, such as muscular dystrophy, sarcopenic frailty of the elderly, cancer-related muscle loss, and ALS (amyotrophic lateral sclerosis), as well as metabolic diseases.
Several F13β2 mRNA fusion libraries (C-NNS, A-50% NNS and A-NNS) were used independently for in vitro selection against biotinylated human myostatin. For each round of selection, DNA from PCR was transcribed by using the MegaScript transcription kit (Ambion). The puromycin-containing linker TEG 6/10 was synthesized and crosslinked to RNA as previously described (Kurz et al, Nucleic Acids Res. 2000, 28:E83). The crosslinked mixture was included in an in vitro translation reaction by using the rabbit reticulocyte lysate translation kit (Ambion) in the presence of 35S-labeled methionine at 30° C. for 60 min. To enhance the fusion formation, 0.5 M KCl and 0.05 M MgCl2 were added to the reaction and incubated for 30 min at 4° C. Next, reactive cysteine residues were modified for 1 hr at room temperature with 10 mM N-ethylmaleimide. Fusion molecules were purified by using oligo dT cellulose chromatography. A reverse transcription reaction was conducted with SuperScript III (Invitrogen) for 1 hr at 45° C. with the primer oligo 65rev (5′-GTCGTCGTCCTTGTAGTCGCGGCGTTGAATCTGCACGTC-3′) (SEQ ID NO:3). Reactive cysteine residues were modified once more for 1 hr at room temperature with 1 mM 2-nitro-5-thiocyanatobenzoic acid or 1 mM N-ethylmaleimide at alternative rounds for the first three rounds of selection. Fusion molecules were further purified by anti-FLAG affinity chromatography on M2 agarose (Sigma) and eluted in buffer A (1×PBS, 0.02% Triton X-100, 1 mg/ml bovine serum albumin [BSA], 0.1 mg/ml salmon sperm DNA [pH 7.4]), containing 100 μg/mL Flag-peptide (DYKDDDDD (SEQ ID NO: 33)). The fusion yield was calculated based on specific activity measured by scintillation counting of 35S-methionine in the samples. Each library contained approximately 1013 variants, which significantly exceeds the diversity of phage display (109), or ribosome display (1011) libraries, providing a larger pool for binder selection. The selections were performed for eight to nine rounds (FIGS. 9A-9C), and lower target concentrations and prolonged washes were utilized in later rounds of selection to favor clones with better affinities. Fusion libraries were incubated separately with 100 μl M-280 beads (Dynal) for 30 minutes at 30° C. prior to selection (preclear). The beads were washed five times with 0.5 ml of buffer A, containing 2 mM D-biotin. The supernatant was then incubated in buffer A with biotinylated human myostatin for 1 hr at 30° C. The target was captured on 100 μl M-280 beads for 10 min at 30° C., and the beads were washed five times with 0.5 ml of buffer A, containing 2 mM D-biotin. Starting from round 2, a third wash was performed for 15 minutes at 30° C. Additional stringency was added to A-50% NNS library selection in R7 and R8: both fusion incubation with the target and third wash were performed for 30 minutes at 30° C. Bound fusion molecules were eluted with 50 μl 0.1 M KOH into 25 μl 1 M Tris-HCl (pH 8.0). DNA was amplified by PCR with flanking primers oligo 55 and oligo 26rev. Primers oligo 77 and oligo 73rev were employed for amplification of Aext-NNS library. Final concentrations of biotinylated human myostatin used in the selections were as follows: R1 (250 nM), R2-R5 (100 nM), R6 (C-NNS and A-NNS: 100 nM; A-50% NNS: 100 and 10 nM); R7 (C-NNS and A-NNS: 100 nM and 10 nM; A-50% NNS: 10 and 1 nM), R8 (C-NNS and A-NNS: 10 nM and 1 nM; A-50% NNS: 1 nM), R9 (C-NNS: 1 nM; A-NNS: 1 and 0.1 nM). Binding to 100 nM of biotinylated human myostatin increased in round 5 (A-50% NNS library) or round 6 (C-NNS and A-NNS libraries). In the next round myostatin concentration was decreased to 10 nM and further to 1 nM to converge the pool for tighter binding to the target. Selections were stopped at round 8 (A-50% NNS library) and round 9 (C-NNS and A-NNS libraries), when no further significant increase in binding to the target was observed. PCR DNA of selection pools after rounds 5, 6, 7, 8 and 9 was cloned into pCR®2.1-TOPO® vector (Invitrogen), and single clones were analyzed by sequencing of 40-150 clones.
The selected protein pools bound to myostatin specifically since no interaction with several unrelated proteins tested at high concentration was observed (FIG. 10). Also, no binding to streptavidin beads was detected, which indicated that the negative selection (preclear) against M-280 beads and an extensive wash with 2 mM biotin were effective in suppressing streptavidin binders.
The sequences of the binding loops are shown in Table 3. Sequence analysis of selected clones revealed that several consensus motifs emerged during the selection. Three major groups could be identified in A-50% NNS library selection pools: group M1-A50 (BC loop—TNPFKETLRS (SEQ ID NO:4), DE loop—REIPPKTT (SEQ ID NO:5), FG loop—RTQYLI (SEQ ID NO:6), V91L); group M2-A50 (BC loop—SNPFKETLRN (SEQ ID NO:7); DE loop—SEIRPNST (SEQ ID NO:8), FG loop—PSLLHL (SEQ ID NO:9)) and group M3-A50 (BC loop—TNPFKETLRN (SEQ ID NO:10), DE loop—CEIRPNSI (SEQ ID NO:11); FG loop—CSLSPW (SEQ ID NO:12), ΔS82, M83R).
Only very few conservative changes were observed in BC loop: T27S, L30F, N36S. Highly hydrophobic mutation L30F was found in nearly all myostatin binding clones, suggesting that it may be beneficial for the binding to the target. The rest of BC loop residues were independently reverse selected to wild type, as indicated by different codon usages in selected clones. Remarkably, wild type BC loop was found in group M1-A binders (BC loop—TNPLKETLRN (SEQ ID NO:13); DE loop—PAIPMNHK (SEQ ID NO:14); FG loop—PQLFHI (SEQ ID NO:15)), which dominated A-NNS library selection. This could probably occur through random recombination event of a binder sequence with wild type clone and further affinity selection. Similar recombination happened when an extended F13β2 library (Aext-NNS) was subjected to selection against myostatin. Wild type BC loop is observed in major group M1-Aext with extended FG loop (BC loop—TNPLKETLRN (SEQ ID NO:16); DE loop—LKIPGC (SEQ ID NO:17); FG loop—MLGLPFSLRFIP (SEQ ID NO:18). This could indicate that the structure of BC loop tolerates only few conservative changes. In this case randomization scheme in A-50% NNS library provides the best opportunity to introduce only few mutations that do not disturb the loop structure. At the same time, it is possible that wild type BC loop is selected, because it has certain affinity to myostatin. Since we didn't observe any binding of wild type F13β2 scaffold to myostatin this is unlikely.
Upon selection DE loop was significantly changed only when NNS randomization was originally applied in A-NNS and Aext-NNS libraries, while reverse selection to wild type residues was observed in A-50% NNS selection pools. This could suggest that DE loop can tolerate many changes but only few are sufficient for binding to myostatin.
Most of the mutations were selected in FG loop: four to six residues were changed out of six randomized positions in A-NNS and A-50% NNS libraries. Several different consensus motifs were selected to bind to myostatin. High variability in FG loop suggests that this part of the scaffold is very tolerant to structural changes and positioned optimal for binding to the target. Indeed, six amino acid insertion into FG loop (group M1-Aext) and single residue deletion and mutation before FG loop (group M3-A50) also resulted in high affinity binding to myostatin. In contrast, when only two residues of FG loop were randomized in conservative (C-NNS) library, the selection yielded molecules with changed BCD β-sheets of the scaffold, as a result of frameshift mutation. These binders partially lost the F13β2 fold and resembled large peptides. This supports the importance of providing a sufficient randomization surface in FG loop, which plays the major role in target binding.
Remarkably, a similar FG loop sequence was identified independently from A-50% NNS (PSLLHL (SEQ ID NO:19)) and A-NNS libraries (PQLFHI (SEQ ID NO:20))—P(S/Q)L(L/F)H(L/I), with 3 identical residues and 3 conservative changes. This could indicate that these molecules bind to the same site on myostatin, which was confirmed later in radioactive binding assay (see below).
Additional loop exchange between clones from different sequence groups (A-50% NNS library) in selection PCR increased the diversity of the pool and allowed for the best combination to be selected (Table 3).







TABLE 3







Selected myostatin binders (grouped by FG loop sequence)



















SEQ

SEQ

SEQ









ID

ID

ID
Scaffold
In vitro
Kd,
B max,
Solubility
Clone


BC loop
NO:
DE loop
NO:
FG loop
NO:
mutation
clone
nM
%
(37/RT/18)
ID










Group M1-A50 (A-50% NNS library)














TNPFKETLRS
4
REIPPKTT
5
RTQYLI
6
V91L
Typical sequence


















TNPFKETLRS
4
REIPPKTT
5
RTQYLI
6
V91L
39633-C1
39.90
74.81
Poor/NA/
M17












YES



TNPFKETLRS
4
REIPPKTT
5
RTQYLI
6
V91L
39633-F5
32.15
69.70










mut
I3Y,













M83I)






TNPFKETLRS
4
REIPPKTT
5
RTQYLI
6
V91P













mut







TNPFKETLQS
34
REIPPKTT
5
RTQYLI
6
V91L







TNPFKETLRG
35
REIPPKTT
5
RTQYLI
6
V91L













mut







TNPFKETLRS
4
RKIPPKTT
81
RTQYLI
6
V91L







TNPFKETLRS
4
RVIPPKTT
82
RTQYLI
6
V91L
39633-C5
30.84
93.05
NO/NA/YES
M34


TNPFKETLRS
4
RVIPPKTT
82
RTQYLI
6
V91L







TNPYKETLRS
36
RVIPPKTT
82
RTQYLI
6
V91L



NO/NA/YES
M83


TNPFKETLRS
4
REIPPETT
83
RTQYLI
6
V91L
39908-F8
36.58
45.71










mut
(K52R)






TNPFKETLRS
4
SEIRPNST
8
RTQYLI
6
V91L
39633-G5
33.57
64.14




SNPFKETLRN
7
SEIRPNST
8
RTQYLI
6
V91L
40636-D2






SNPFKETLRN
7
REIPPKTT
5
RTQYLI
6
V91L
39633-G1
38.90
52.00




SNPFKETLRN
7
RVIPPKTT
84
RTQYLI
6
V91L



Poor/NA/
M3












YES



TNPLSQTLSA
37
REIPPKTT
5
RTQYLI
6
V91L
39633-B3
9.93
6.14




SNPFKETLRN
7
CEIRPNSI
11
RTQYLI
6
V91L
39633-F4
26.75
28.47




SNPFKETLRN
7
CEVRPNSV
85
RTQYLI
6
V91L
39633-E2
27.28
20.99









Group M2-A50 (A-50% NNS library)














SNPFKETLRN
7
SEIRPNST
8
PSLLHL
9

Typical sequence


















SNPFKETLRN
7
SEIRPNST
8
PSLLHL
9

39908-G4
28.36
48.95
Poor/NA/
M7












YES



SNPFKETLRN
7
SEIRPNST
8
PSLLHL
9
mut
39633-D3
26.19
65.77
NO/NO/NA










(V39M)






SNPFKETLRN
7
SEIRPDST
86
PSLLHL
9

39633-E4
27.75
59.99
NO/NO/YES
M019


SNPFKETLRN
7
CEIRPNSI
11
PSLLHL
9








TNPFKETLRS
4
SEIRPNST
8
PSLLHL
9

39908-G12
25.59
34.11
Poor/NA/
M10












YES



TNLMKETLRN
38
SEIRPNST
8
PSLLHL
9

39633-D2
11.15
6.74




TNPFKETLRS
4
REIPPKTT
5
PSLLHL
9

39908-H10
29.73
47.28
Poor/NA/
M4












YES



TNPFKETLRS
4
REIPPKTT
5
PSLLHL
9
mut
39633-H6
24.83
61.65











(M50V)











Group M3-A50 (A-50% NNS library)














TNPFKETLRN
10
CEIRPNSI
11
CSLSPW
12
ΔS82, M83R
Typical sequence


















TNPFKETLRN
10
CEIRPNSI
11
CSLSPW
12
ΔS82, M83R
39633-D6
20.26
79.62
Part/NA/NA
M020


TNPFKETLRN
10
CEIRPNSI
11
CSLSPW
12
ΔS82, M83R













mut







SNPFKETLRN
7
CEIRPNSI
11
CSLSPW
12
ΔS82, M83R
39908-A1
12.01
50.22
Part/NA/NA
M02


SNPFKETLRN
7
CEIRPNSI
11
CSLSPW
12
ΔS82, M83R



Part/NA/NA
M013








mut







SNPFKETLRN
7
CEIRPNSI
11
CSLSPW
12
ΔS82, M83K







SNPFKETLRN
7
CEIRPNST
87
CSLSPW
12
ΔS82, M83R













ΔI8







SNPFKETLRN
7
CGIRPNSI
88
CSLSPW
12
ΔS82, M83R







SNPFKETLRN
7
CEVRPNSI
89
CSLSPW
12
ΔS82, M83R
39633-A5
19.36
78.35










mut
(V46I)






SNPFKETLRN
7
CEIRPNSI
11
CSLPPW
150
ΔS82, M83R







SNPFKETLRS
39
CEIRPNSI
11
CSLSPW
12
ΔS82, M83R













mut







TNPFKETLRS
4
CEIRPNSI
11
CSLSPW
12
ΔS82, M83R
39908-C6
19.66
52.18
NO/NA/YES
M05


TNPFKETLRS
4
CEIRPNSI
11
CSLSPW
12
ΔS82, M83R













mut







TNPFKETLRS
4
CEIRPNRI
90
CSLSPW
12
ΔS82, M83R
39908-A3
11.03
57.18
NO/NA/Poor
M09


TNPFKETLRN
10
SEIRPNST
8
CSLSPW
12
ΔS82, M83R













mut







TNPFKETLRN
10
SEIRTNAT
91
CSLSPW
12
ΔS82, M83R







SNPFKETLRN
7
SEIRPNST
8
CSLSPW
12
ΔS82, M83R
39908-E5
21.88
77.85
YES/NA/YES
M56


SNPFKETLRN
7
SEIRTNAT
91
CSLSPW
12
ΔS82, M83R
39908-C11
16.96
73.74
NO/NA/YES
M22


SNPFKETLRN
7
SEIRPNSI
92
CSLSPW
12
ΔS82, M83R













mut







SNPFKETLRN
7
RDIRPKTK
93
CSLSPW
12
ΔS82, M83R



NO/NA/YES
M58


TNPFKETLRS
4
REIPPKTT
5
CSLSPW
12
ΔS82, M83R
39908-D2
23.34
68.83
Part/NA/NA
M29


TNPFKETLRS
4
REIPPKTT
5
CSLSPW
12
ΔS82, M83R













mut







TNPFKETLRS
4
SEIRPNST
8
CSLSPW
12
ΔS82, M83R



YES/NA/YES
M71


TNPFKETLRS
4
RDIRPKTK
93
CSLSPW
12
ΔS82, M83R







TNPFKETLRS
4
SEIRTNAT
91
CSLSPW
12
ΔS82, M83R













mut







TNPFKETLRS
4
REIPPETT
94
CSLSPW
12
ΔS82, M83R













mut







TNPFKGTLRS
40
WEIPPKTT
95
CSLSPW
12
ΔS82, M83R













mut







TNPFKETLRN
10
RDIRPKTK
93
CSLSPW
12
ΔS82, M83R
39908-A2
15.21
68.49
NO/NO/YES
M03


TNPFKETLRN
10
CEIRPNSI
11
CQLLPW
157
ΔS82, M83R
39908-F5
27.07
67.24
YES/NA/NA
M16


SNPFKETLRN
7
CEIRPNSI
11
CQLLPW
157
ΔS82, M83R













mut







SNPFKETLRN
7
SEIRPNST
8
CQLLPW
157
ΔS82, M83R













mut







SNPFKETLRN
7
RDIRPKTK
93
CQLLPW
157
ΔS82, M83R
39908-H4
15.80
68.58
NO/NO/YES
M015


SNPFKETLRN
7
RDIRPKTK
93
CQLLPW
157
ΔS82, M83R













mut







SNPFKETLRN
7
REIPPKTT
5
CQLLPW
157
ΔS82, M83R













mut







TNPFKETLRS
4
REIPPKTT
5
CQLLPW
157
ΔS82, M83R
39908-B2
27.10
75.86
Part/YES/
M07












YES



TNPFKETLRS
4
REIPPKTT
5
CQLLPW
157
ΔS82, M83R













mut







TNPFKETLRS
4
RDIRPKTK
93
CQLLPW
157
ΔS82, M83R













mut







TNLMKETLRN
41
RDIRPKTK
93
CQLLPW
157
ΔS82, M83R







TNPWAETLHN
42
RDIRPKTK
93
CQLLPW
157
ΔS82, M83R







SNPFKETLRN
7
CEIRPNSI
11
-TLRPW
152




NO/Poor/NA



SNPFKETLRN
7
CEIRPNSI
11
MTLRPW
153
ΔS82, M83R
39908-A7
20.14
109.40
Part/NA/NA
M24


SNPFKETLRN
7
CEIRPNSI
11
-ALRPW
154
V69A







SNPFKETLRN
7
REIATMST
96
-TLRPW
152








TNPFKETLRS
4
REIPPKTT
5
-TLRPW
152

39633-G6
19.69
75.13
No exp/NA/
M18












No exp



TNPFKETLRS
4
REIPPKTT
5
-TLRPW
152
mut







TNLWKETLRN
43
REIATMST
96
-TLRPW
152








TNLWKETLRN
43
REIATMST
96
-TLRPW
152
mut







TNLWKETLRN
43
REIATMST
96
-TLRPW
152
mut







TNLWKEALRN
44
REIATMST
96
-TLRPW
152
V39M
39908-E1
14.85
40.22
No
M04












exp/NO/NA



TNLWKETLRN
43
REIPPMST
97
-TLRPW
152
mut












Group M4-A50 (A-50% NNS library)


















TNPFKETLRS
4
REIPPKTT
5
CEPSLD
155

39633-
8.69
6.91











D5/yes






TNPFKETLRS
4
REIPPKTT
5
YVAPLH
156




YES/NA/NA
M21


TNPFKETLRS
4
REIPPKTT
5
GTQFPR
157
mut







TNPFKETLRS
4
REIPPKTT
5
PSDSLP
158
mut







TNPFKETLRS
4
REIPPNTT
98
PTHHLL
159

39633-A1
25.52
63.94




SNPFKETLRS
45
EEIRQSCI
99
RD-ALS
160
mut







SNPFKETLRN
7
CEIRPNSI
11
GSDCLG
161
mut







TRPLNDRLRK
46
REIRPTSS
100
NTESIL
162
mut







TTPQEETLIK
47
MKIPPASW
101
MSDTLR
163








VTPLWANLCT
48
WDIRHDPT
102
SKPPER
164
mut












Group M1-A (A-NNS library)













TNPLKETLRN
13
PAIPMNHK
14
PQLFHI
15
Typical sequence


















TNPLKETLRN
13
PAIPMNHK
14
PQLFHI
15

39885-A1
21.76
67.26
NO/NO/Part
M1-1


TNPLKETLRN
13
PAIPMNHK
14
PQLFHI
15
mut
39885-
25.14
72.16
No exp/NA/
M2-1









B5(F26V)


Poor










39885-
15.43
73.68
NA/NA/No










F10(S82C)


exp










39888-
13.55
82.36
NA/NA/Very
M14-1









All(D42N)


Poor










39888-
9.988
83.12











G5(S82I)






TNPMKETLRN
49
PAIPMNHK
14
PQLFHI
15








TNPLKEILRN
50
PAIPMNHK
14
PQLFHI
15








TNPLRETLRN
51
PAIPMNHK
14
PQLFHI
15








TNPLKETLRN
13
RAIPMNHK
103
PQLFHI
15

39885-C7
17.43
68.37
NO/NA/Poor
M3-1


TNPLKETLRN
13
PTIPMNHK
104
PQLFHI
15

39885-G8
30.75
69.21
NA/NA/Poor
M10-3


TNPLKETLRN
13
PTIPMNHK
105
PHLFHI
165

39885-F7
24.11
62.72




TNPLKETLRN
13
PAIPYLSY
106
PQLHHL
166

39885-G5
28.49
59.43
NA/NA/YES
M8-L


TNPLKETLRN
13
PAIPMNHK
14
PQLFHL
167

39885-F8
28.1
98.61
NO/NA/YES
M7-L


TNPLKETLRN
13
PAIPMNHK
14
PQLFHL
168
mut







TNPLKETLRN
13
PAIPMNHK
14
PRLFHI
169
V39M







TNPLKETLRN
13
PAIPMDHK
107
PQLFHI
15

39885-
24.73
57.62
NO/NO/Poor
M5-2









D8(K9R)






TNPLKETLRN
13
PTISRFKR
108
PQLQHL
170

39973-C10
22.79
71.98
NO/NO/No













exp



TNPLKETLRN
13
ATIPPYLP
109
PQLIHL
171

39973-F10
28.65
68.02
NO/NA/NO



TNPLKETLRN
13
SLICVNRA
110
PQLFHI
15













Group M2-A (A-NNS library)


















TNPLKETLRN
13
PAIPMNHK
14
INYFYK
172








TNPLKETLRN
13
PAIPMNHK
14
INYFYK
172

39885-C11
41.59
18.77




TNPLKETLRN
13
PAIPMNHK
14
INYFYK
172
mut



NO/NO/NA



TDPLKETLRN
52
PAIPTNHK
111
INYFYK
172
mut







TNPLKETLRN
13
PAIPMNHK
14
INHFYK
173

39885-D11
41.03
22.26




TNPLKETLRN
13
PAIPMNHK
14
INYFYN
174

39885-H9
14.14
24.06




TNPLKETLRN
13
PAIPMKHK
112
LNHFYK
175
H76Y, E94G







TNPLKETLRN
13
PAIPMNHK
14
LNHFYK
176

39885-C1
140
33.34









Group M3-A (A-NNS library)


















HSMSSGILNY
53
DLIRNSHV
113
HHGSYP
177








YFSPGARLAT
54
HSIYVPFW
114
TTTVQG
178

39973-D2
32.89
7.656




KHDRYRYLQQ
55
KLIIFSYP
115
RFMCPS
179

39973-B12
34.54
7.77




AATLYHALFS
56
LNISAQTM
116
CHAMTS
180








RSLPAAWLEN
57
TRIKCASS
117
KVLTEH
181








GLIGLLQLYE
58
CGIPPLSS
118
YLELLY
182








PCMGQYKLPL
59
MEITINGY
119
RHTDWS
183








LIGILCL-WY
60
MQILLYIL
12
QHSPPA
184
Q14R







ARTIHTILTN
61
TMISWNLN
121
SAGCSF
185
Q64R







MNIIRNMLTT
62
IMIDKLML
122
QTAEPR
186
E66G







QTKLIQSLVT
63
HRIPCLT-
123
SLLITP
187
S82N







FHTYRLWLGV
64
WLISNIDL
124
VYIKNG
188
PG (44-













45)/MLADW







TNPLKETLRN
13
EQIRESEY
125
LTETRP
189

39973-G3
21.97
1.806




TNPLKETLRN
13
PAIPMNHK
14
NSSSNQ
190

39888-B7
7.092
1.189
















TNPLK---ETLRN
16
LKIPGCST
126
SMLGLPFSLRFIP
191

Typical sequence


















TNPLK---ETLRN
16
LKIPGCST
127
SMLGLPFSLRFIP
191

45927-B3
86.70
50.68
NO/NA/YES
Mext


TNPLK---ETLRN
16
LKIPGCST
128
SMLGLPFSLRFIP
191
mut
45927-B7
57.61
46.35











(M50T)






TNPLK---EALRN
65
LKIPGCST
129
SMLGLPFSLRFIP
191








TNPLE---ETLRN
66
LKIPGCST
130
SMLGLPFSLRFIP
191








TNPLK---ETPRN
67
LKIPGCST
131
SMLGLPFSLRFIP
191








TNPLK---ETLRN
16
LKIPGCST
132
SMLGLPFSLRLIP
192








TNPLK---ETLRN
16
QKIPGCST
133
SMLGLPFSLRFIP
191

45927-
65.05
41.22











A11






TNPLK---ETLRN
16
LKILGCST
134
SMLGLPFSLRFIP
191

45927-A3
53.16
36.38




TNPLK---ETLRN
16
TGILQWST
135
SPHAWWQHHGNFS
193

45927-E1
6.04
4.57




TNPLK---ETLRN
16
LKIPGCST
136
SMRMNLLFHLMNA
194








TNPARGVNPSLRN
68
TDICKGST
137
SYNVCRRVLKSFI
195
W65G,













V91E







TNPFALTCRNLRN
69
SSINIFST
138
SLASAAPMMIQTW
196








TNPLTNWGVWLRN
70
NAITPVST
139
SCGLYGSQCCYTW
197








TNPNHFTRRELRN
71
MNIAAEST
140
SHLFSDDLWAAPT
198








TNPSCSSHCSLRN
72
TWIWLYST
141
SLLGWPMNVTLKR
199








TNPTDGSRPWLRN
73
FLIRTDST
142
SYGRCMVNLVRPL
200








TNLTNTHYSRLRN
74
QAITSCST
143
STSDSGWASNIVP
201
P29L







TNPARGKTMNLRN
75
MIIYTNST
144
SCKLDQNCSALNL
202
Δ11-13













Δ44-45







TNPGSECNLSLRN
76
LYITPGST
145
SRYKLVVLARKVG
203








TNPDAWAKCVLRN
77
SGIASLST
146
SSLICRVKDFWMD
204








TNPKRDTSVWLRN
78
FTIGYPST
147
SPSILSKGNIGLG
205
H76R
45927-
16.05
8.07











A10






TNPLRVSEQTLRN
79
GGIRVHST
148
SYVAEYGEWTHYS
206
P47L
45927-
1.47
3.08











A12






TNPSNYRPCILRN
80
KMIVYHST
149
SKFELSGTSSRGR
207

45927-E5
64.07
8.36





Amino acids mutated from wild type after selection are underlined and marked in bold.






Example 6
Measurement of Affinity of Selected Myostatin ETBPs In Vitro
Affinities of selected F13β2 binders to biotinylated myostatin were determined in radioactive equilibrium binding assay (FIGS. 11A and 11B; Table 3). 35S-labeled binder proteins from selection pools and single clones were produced in vitro in a rabbit reticulocyte lysate translation kit (Ambion) and purified on M2-agarose (Sigma). Varying concentrations of biotinylated human myostatin (0-100 nM) or other proteins (protein 1: 100 nM; peptide 1: 250 nM; protein 2: 50 nM) were incubated with a constant concentration of the purified binder protein (1 or 2 nM) at 30° C. for 30 min in buffer B (1×PBS, 0.02% Triton X-100 [pH 7.4]). The receptor-binder complexes were captured by using 25 μl of M280 or Protein A (Dynal) (protein 2) magnetic beads for 10 min at room temperature on a Kingfisher instrument (Thermo Fisher Scientific). The beads were washed five times with 100 μl buffer B, and the amount of 35S-methionine remained on the beads was measured by scintillation counting. Data were analyzed by using the GraphPad Prism software (GraphPad Software, Inc., San Diego, Calif.), fitted with a one-site, nonlinear binding equation. For inhibition assay the binding of 35S-labeled proteins to 20 or 25 nM of biotinylated human myostatin was performed in the presence of varying concentrations of inhibitor (human activin receptor IIB/Fc chimera (R&D Systems, Minneapolis, Minn.) (0-250 nM); protein 1 (100 nM), peptide 1 (250 nM), protein 2 (50 nM); proteins, produced in E. coli, M17, M7, M29, M21, M1-1, WT F13β2 (500 nM)).
Moderate affinity binders from group M1-A50 and group M2-A50 interacted with myostatin with Kd of 31-40 nM and 25-30 nM, respectively. Conservative scaffold mutations in selected clones (ex., F26V, V39M, V46I, M50V, M83I) did not affect the binding affinity of the proteins. Also, no change in binding was observed when the wild type residue persisting in the DE loop was mutated (E56V), supporting the idea that the reverse selected wild type residues do not participate in binding to the target. In contrast, mutation of the consensus lysine residue in the DE loop (K60E) results in a decrease of binding activity, indicating that this position affects interaction with the target. Proteins from group M3-A50 and group M1-A bind myostatin with higher affinities (Kd of 11-27 nM and 10-31 nM, respectively). Some variability in the FG loop does not affect the binding: neither deletion of C84 and mutations C84M, S85Q/T and S87UR in the group M3-A50 proteins, nor mutations Q85H/R, F87H/Q/I, 189L in the group M1-A binders change the interaction with myostatin. On the other hand, exchange of selected FG loop sequences (but not DE loop sequences, group M1-A) to unrelated sequences significantly decreases the binding, indicating a major role for FG loop residues in interaction with the target (group M2-A, group M3-A). Conservative residues that are present in the FG loop of all highly potent binders (L86, P88, W89 in group M3-A50, and P84, L86, H88 in group M1-A) potentially form the binding site with myostatin. The conserved phenylalanine in the BC loop of the binders selected from A-50% NNS library appears necessary for interaction with myostatin, since even modest changes to this residue abolish (F20L/M) or reduce (F20W) the binding to the target (FIGS. 11A and 11B). Remarkably, the BC and DE loops of ETBPs from group M1-A50, group M2-A50 and group M3-A50 can be interchanged without significant alteration in the binding activity, which suggests that they may bind to the same site on the myostatin molecule. Binders selected from Aext-NNS library showed lower affinity (Kd=53-86 nM) for the target, which was also correlated with a decreased B max (Table 3). Binders from this group have an extended highly hydrophobic FG loop (MLGLPFSLRFIP (SEQ ID NO:21)), and it is possible that the observed decreased affinity is related to poor biophysical properties of the proteins.
Highly potent binders from group M3-A50 and group 1-A were observed among sequenced isolates from selection under stringent conditions (FIGS. 12A and 12B): in earlier rounds low affinity binders were found to dominate the selection pool, but by the last round of selection the high affinity binders constituted more than 80% of all proteins.
Myostatin binder proteins produced in E. coli could efficiently inhibit the interaction between myostatin and various in vitro-produced myostatin binders (FIG. 13A). Wild type F13β2 domain did not compete for binding. These data indicate that selected proteins from the four major groups bind to similar sites on the myostatin molecule.
The best binders from different sequence groups were subsequently tested for binding to myostatin in the presence of human activin receptor IIB (ARIIB) (FIG. 13B). Activin receptor efficiently inhibited target binding of the proteins with moderate affinities to myostatin—M17 (group M1-A50) and M7 (group M2-A50), suggesting that these binders may interfere with the natural ARIIB-myostatin interaction. Only a modest inhibition (20%) of target binding of high affinity proteins from group M3-A50 (M29) and group M1-A (M1-1) was observed at the highest concentration of activin receptor (250 nM). Since the selected binders bind to similar sites on myostatin (see above), it is possible that in vitro binding assay conditions are more favorable to binding of highly potent F13β2 proteins than activin receptor, and higher inhibitor concentrations would be needed to efficiently displace the binder from myostatin surface.
Example 7
Production and Purification of Selected Myostatin ETBPs from E. Coli 
DNA from myostatin binder clones was amplified by PCR with primers oligo 53 and oligo 28rev to introduce NdeI at 5′ end, and His6-tag-Stop (“His6” disclosed as SEQ ID NO: 29) sequence and Barn HI site at 3′ end, respectively. To generate proteins, containing PSM-His6-Stop (“His6” disclosed as SEQ ID NO: 29) (Barn HI site) and PSM-Stop (XhoI site) sequences oligo AKx198 and oligo AKx299 were used for 3′ end in PCR. To construct F13β2-GFP fusions a primer oligo 18rev (Barn HI) was used for 3′ end in PCR which allowed introduce the binder proteins at the N-terminus of GFP. The binder proteins were expressed in E. coli by using a modified pet28(a+) vector. Protein expression was induced with 0.25-1 mM IPTG for 3 hours at 37° C. or overnight at room temperature or 18° C. Cell pellets were collected, and after freeze/thaw disruption, resuspended in 1×PBS (pH 7.4), containing LysonaseTM Bioprocessing Reagent (EMD Chemicals Inc., San Diego, Calif.). After incubation at room temperature for 20 minutes CHAPS was added to 0.4% final concentration. The solution was rotated for 20 minutes at 4° C., and the soluble fraction was separated by centrifugation. Aliquots of total and soluble fractions were collected before and after centrifugation for SDS-PAGE analysis. When F13□2-GFP fusion proteins were expressed the fluorescence was monitored under UV light in cell pellet, insoluble pellet and soluble fraction. For His-tag affinity purification the supernatant was rotated for 1 hr at 4° C. with Ni-NTA agarose (Qiagen) preequilibrated with buffer C (1×PBS [pH7.4], 0.5 M NaCl; 20 mM Imidazole, 0.3% CHAPS). The resin was washed with 50 column volumes of buffer C and 30 column volumes of buffer D (1×PBS [pH7.4], 20 mM Imidazole). Protein was eluted with 1×PBS, 200 mM Imidazole (pH 7.4) and was dialyzed against 1×PBS at 4° C.
The solubility in physiological conditions (1×PBS, pH 7.4) varied between different binders (Table 3, FIG. 14A) when expressed at 37° C. and RT, but all binders were soluble to some extent when expressed at 18° C. The best solubility profile was observed for binders from group M3-A50, for which the majority of the proteins were soluble in PBS after expression at 37° C. We purified a number of myostatin binders from the soluble fraction by affinity chromatography via a His6 tag (SEQ ID NO: 29) yielding 5-20 mg of each protein from one liter culture. E. coli-produced binders were able to compete for binding to myostatin in a radioactive equilibrium binding assay in vitro (see above), and their activity was further tested in a cell proliferation assay.
To analyze the folding pattern of different myostatin binders we expressed them as N-terminal fusions with GFP as a “folding reporter”. Since folding and formation of the GFP chromophore has been reported to depend on the correct folding of the upstream protein we could assess the folding of the myostatin binders by GFP fluorescence and compare it with the solubility profile in PBS (FIG. 14B). Myostatin binder M29 from group M3-A50 was soluble in PBS, as shown by SDS-PAGE. We could monitor the disappearance of green fluorescence in the insoluble pellet after solubilization, which indicated that the majority of correctly folded M29-GFP protein was in the soluble fraction. The M7 binder from group M2-A50 remained in the insoluble pellet, but was correctly folded, as indicated by a strong GFP signal. Since the majority of the E. coli proteins are found in the soluble fraction (FIG. 14B); the pellet is 80 to 90% enriched with a binder protein.
To apply this finding for the purification of the binder proteins we expressed various myostatin binders at 37° C. to achieve the highest expression. Cell pellet was resuspended in PBS diluted (1:5) with water and incubated with LysonaseTM Bioprocessing Reagent at room temperature for 20 minutes. Following incubation with 0.33% CHAPS the soluble fraction was separated by centrifugation. Since F13β2 proteins that remained in the pellet are correctly folded no re-folding procedure was necessary. After testing a number of different conditions we found that 36 mM HCl was optimal for solubilization of the correctly folded proteins. Any remaining insoluble material was removed by centrifugation and the supernatant was dialyzed against water. The myostatin binders were analyzed by SDS-PAGE (FIG. 15). Presence of His6 tag (SEQ ID NO: 29) or PSM sequence on the C-terminus of the myostatin binder protein (M56-His6 (“His6” disclosed as SEQ ID NO: 29), M56-PSM-His6 (“His6” disclosed as SEQ ID NO: 29), M56-PSM) (FIG. 15) didn't change the extraction behavior although production and solubility of protein which contained only PSM sequence on the C-terminus was higher. The enrichment of F13β2 proteins achieved in a single extraction step ranged from 80 to 90% and was successfully applied for purification of tagless myostatin binders. This simple purification procedure of F13β2 binders can be utilized for high throughput screening of a large number of nearly pure proteins and ultimately gives a great advantage for therapeutic applications.
The thermostability of the myostatin binding proteins derived from F13β2 scaffold differed from binder to binder, and Tm values ranged from 55° C. to 70° C. at neutral pH.
Example 8
Measurement of Biological Activity of Selected Myostatin ETBPs
The biological activity of F13β2 myostatin binders was tested by an MPC-11 cell proliferation assay. Mouse myeloma MPC-11 cells (ATCC#CCL-167, American Type Culture Collection, Manassas, Va.) were grown in Dulbecco's modified Eagle's medium (DMEM; Invitrogen), supplemented with 10% (v/v) horse serum, and 0.05 mg/ml gentamicin. For cell proliferation assay, serial dilutions of human activin A (Peprotech (Rocky Hill, N.J.)) (0-100 ng/ml), GDF-11 (Peprotech (Rocky Hill, N.J.)) or myostatin (Peprotech (Rocky Hill, N.J.)) (0-1000 ng/ml) (100 μl) were incubated in growth medium with 2000-4000 cells/well in 96-well tissue culture plate. For inhibition assay, the cells were resuspended in growth medium, containing 10 ng/ml of activin A, or 125-250 ng/ml of GDF-11 or myostatin, and 170 μl containing 2000-4000 cells were added per well to a 96-well tissue culture plate. Serial dilutions of F13β2 proteins in 1×PBS (pH 7.4) (30 μl) were added to each well, and the cells were incubated for 72 hours at 37° C. Cell proliferation was measured by the addition of 10 μl CCK-8 solution (Cell Counting Kit-8; Dojindo Molecular Technologies, Inc., Gaithersburg, Md.) to each well, followed by incubation for 8 hr at 37° C., and measurement of the absorbance at 450 nm with a microtiter plate reader (Molecular Dynamics).
Recombinant human activin A, GDF11 and myostatin inhibit cell proliferation of mouse myeloma cells (MPC-11) in a dose-dependent manner (FIG. 16). F13β2 myostatin binding proteins from different sequence groups were found in the MPC-11 cell assay to antagonize the biological function of myostatin (FIGS. 17A-17D). Several binders from group M1-A50 (M3, M34), group M2-A50 (M7, M019, and M10) and group M1-A (M1-1, M2-1, and M3-1) showed inhibition activity at high concentrations. The binders from group M3-A50 (M56, M71, and M07) neutralized the myostatin activity in a dose-dependent fashion with better affinities (IC50s as low as 150-200 nM). The potency of inhibition appeared to be ten times lower than that of activin receptor IIB. The difference in potency in biological assay correlated with the affinity to the target and biophysical properties of the proteins: binders from group M3-A50 are highly potent against myostatin and have good biophysical properties as shown by their solubility profile. The presence of a cysteine residue in DE loop of group M3-A50 binders (M020, M02, M05, and M16) decreased their biological activity. It is possible that additional cysteine destabilized the proteins during the assay (conducted at 37° C. over 3 days) and/or formed nonspecific complexes with other proteins in the reaction media. The activity of tagless F13β2 proteins purified by acidic conditions was tested in the MPC-11 proliferation assay in which they also showed inhibition activity towards myostatin. This indicates that the acidic extraction procedure allows us to obtain fully functional F13β2 binder proteins. Additionally, the selected myostatin binders antagonized the activity of GDF11, another member of TGFβ family, which has high sequence identity to myostatin (89.9%). However, no inhibition of activin A biological action on MPC-11 cells was observed with myostatin binders. Activin A is distantly related to myostatin (sequence identity of 33.3%) and binds to the same receptors—activin receptor IIA and activin receptor IIB. This indicates that the binders are selective for myostatin and closely related proteins, but do not recognize distant members of the same family.
Example 9
Characterization of Pegylated Myostatin ETBP
For application in vivo, fusions of therapeutic proteins with other proteins, peptides or polyethylene glycol are widely used to produce molecules with longer half-life; in addition PEG-substituted proteins are known to exhibit reduced immunogenicity. One advantage of using a small scaffold protein that does not have a structural requirement for free cysteines is the possibility of introducing one or more surface-exposed cysteines to facilitate substitution with prosthetic groups, crosslinking to other proteins, or substitution with polymers such as linear or branched polyalkylene glycols.
To introduce a single cysteine into the C-terminus of myostatin binder DNA from myostatin binding clone F10 was amplified by PCR with primers oligo 53 and oligo AKx226 to introduce NdeI site at 5′ end, and PCM-Stop sequence and XhoI site at 3′ end, respectively. The binder protein was expressed in E. coli by using a modified pet28(a+) vector. Protein expression was induced with 0.25 mM IPTG for 3 hours at 37° C. and the insoluble pellet was dissolved using acidic conditions as described above for tagless myostatin binding proteins. The protein was pegylated with Y-MAL-40K (Y-Shape PEG Maleimide, JenKem Technology USA Inc., Allen, Tex.) (FIG. 18A). The pegylation reaction was performed at 4° C. under argon in 1×PBS, 2 mM EDTA using protein concentration of 1.5 mg/ml and 4:1 molar ratio of Y-MAL-40K and protein. After 20 hours the protein was dialyzed into 1×PBS, pH 7.4, and analyzed by SDS-PAGE (FIG. 18B). The activity of the pegylated F10 binder in MPC-11 cell bioassay was similar to the free protein.
Example 10
Production of ETBP Dimers in E. Coli 
Dimerization or multimerization of binder molecules separated by various linker sequences can potentially increase binding activity towards a target protein by increases in avidity. Dimerized or multimerized molecules may also have more favorable serum persistence characteristics by virtue of their ability to escape renal filtration. To explore these possibilities we constructed a homodimer of selected myostatin binder E3.
DNA from myostatin binder clone E3 was amplified by PCR with primers oligo 53 and AKx169 to introduce NdeI at 5′ end, and PSM-His6-tag-Stop sequence (“His6” disclosed as SEQ ID NO: 29) and XhoI site at 3′ end, respectively. PCR product was cloned into modified pet28(a+) vector. To introduce a second E3 domain into expression vector, DNA from myostatin binder clone E3 was amplified by PCR with primers AKx202 and AKx203 to introduce XbaI site, an initiator methionine codon and glycine codon at 5′ end, and NNSNNSNNSNNSNNSNNS linker (SEQ ID NO: 208) sequence and NdeI site at 3′ end, respectively. The PCR product was cloned into XbaI and NdeI sites of modified pet28(a+) vector containing E3 clone, described above. The configuration of the dimer molecule is shown in FIG. 19A.
Approximately 100 independent clones were analyzed by expression in E. coli BL21 (DE3) pLysS cells (Invitrogen). Protein expression was induced with 0.25 mM IPTG for 3 hours at 37° C. and the protein was solubilized at acid pH from the insoluble pellet as described above for tagless myostatin binding proteins. Total, soluble and extraction fraction were analyzed by SDS-PAGE (FIG. 19B). Approximately 0.1 mg of protein was produced from 1 ml of culture for different E3 homodimers. When the activity of high-producing E3 homodimer proteins was analyzed in MPC-11 cell proliferation assay they exhibited improved biological activity compared to the single domain.
Example 11
Selection of ETBPs Against Human Nerve Growth Factor (β-NGF)
Nerve growth factor, the founding and best-characterized member of the neurotrophin family, plays a central role in the development, maintenance and survival of the peripheral and central nervous system. It has been proposed as a therapeutic target molecule in many pathological states such as Alzheimer's disease, nervous system injuries, inflammatory or neuropathic pain states, several human malignancies and asthma.
F13β2 mRNA A-50% NNS fusion library was used in in vitro selection against biotinylated human β-NGF. Fusion production and selection procedure was performed as described above for myostatin selections. The selection was performed for nine rounds (FIG. 20), and lower target concentrations and prolonged washes were utilized in later rounds of selection to favor clones with better affinities. Starting from round 2 third wash was performed for 15 minutes at 30° C. From round 3 fusion incubation with the target and third wash were performed for 30 and 45 minutes at 30° C., respectively. Final concentrations of biotinylated human β-NGF used in the selections were as follows: R1 (250 nM), R2-R8 (100 nM), R7-9 (10 nM); R9 (1 nM). After the NGF concentration was decreased to 10 nM in round 7 the specific binding to 100 nM of the target increased in round 8. Selection was stopped at round 9 following target decrease to 1 nM. PCR DNA of selection pools after rounds 6, 8 and 9 was cloned into pCR®2.1-TOPO® vector (Invitrogen), and single clones were analyzed by sequencing of 32-96 clones.
The sequences of the binding loops are shown in Table 4. Several consensus motifs were selected under stringent conditions in rounds 8 and 9: group N1 (BC loop—TNPLNEPLLF (SEQ ID NO:22), DE loop—RIISPHAT (SEQ ID NO:23), FG loop—SIGFQA (SEQ ID NO:24)); group N2 (BC loop—INTSNETLPS (SEQ ID NO:25); DE loop—PEIPPNSS (SEQ ID NO:26), FG loop—IRDSSR (SEQ ID NO:27), Δ67-70, P71A) and group N3 (BC loop—TNLSNETLGT (SEQ ID NO:28), DE loop—PHIRPKVT (SEQ ID NO:30); FG loop—LIASLR (SEQ ID NO:31)). Group N2 binders have a four amino acid deletion at the end of E β-sheet and the beginning of EF loop. Additional mutation of proline to alanine (P71A) in this region probably adds more flexibility to the EF loop and this part of the scaffold. This change could possibly shift the DE loop and expose the BC loop binding site. Indeed, BC loop in this group of binders carriers majority of mutations (6 out 9 residues are changed) compare to the other two loops (in both DE and FG loops 6 amino acids are mutated) and may play the major role in binding to the target.







TABLE 4







Selected β-NGF binders (grouped by FG loop sequence)

















SEQ

SEQ

SEQ







ID

ID

ID
Scaffold
In vitro
Kd,
B max,


BC loop
NO:
DE loop
NO:
FG loop
NO:
mutation
clone
nM
%










Group N1














TNPLNEPLLF
22
RIISPHAT
23
SIGFQA
24

Typical sequence
















TNPLNEPLLF
22
RIISPHAT
23
SIGFQA
24

61362-C6
24.0
5.5


TNPLNEPLLF
22
RIISPHAT
23
SIGFQA
24
mut





TNPLNEPLLF
22
RIISPHAT
23
SVGFQA
288
mut





TNPLSEPLLF
209
RIISPHAT
23
SIGFQA
24
mut





TNPLNDPLLF
210
RIISPHAT
23
SIGFQA
24
mut





TNPLNEPLFF
211
RIISPHAT
23
SIGFQA
24






TNPLNEPLLF
22
PIISPHAT
253
SIGFQA
24






TNPLNEPLLF
22
RIITPHAT
254
SIGFQA
24
mut





FNAQTEALRD
212
RIISPHAT
23
SIGFQA
24
mut

















INTSNETLPS
25
PEIPPNSS
26
IRDSSR
27
Δ67-70, P71A
Typical sequence
















INTSNETLPS
25
PEIPPNSS
26
IRDSSR
27
Δ67-70, P71A
61362-F2
62.0
35.4


INTSNETLPS
25
PEIPPNSS
26
IRDSSR
27
Δ67-70, P71A,











mut





INTSNETLPS
25
PEIPPNSS
26
IRDSSR
27
Δ67-70, P71T,











mut





INTSNETLPS
25
PEIPPNSS
26
IRDSSR
27
Δ67-70, P71S,











mut





INTSNETLPS
25
PEIPPNSS
26
IRDSSR
27
Δ67-70, P71V,











mut





TNTSNETLPS
213
PEIPPNSS
26
IRDSSR
27
Δ67-70, P71A,











mut





TNLSNETLPS
214
PEIPPNSS
26
IRDSSR
27
Δ67-70, P71A,











mut





TNTTNETLPS
215
PEIPPNSS
26
IRDSSR
27
Δ67-70, P71A,











mut





INTSDETLPS
216
PEIPPNSS
26
IRDSSR
27
Δ67-70, P71A





INTSNKTLPS
217
PEIPPNSS
26
IRDSSR
27
Δ67-70, P71A,











mut





INTSNEALPS
218
PEIPPNSS
26
IRDSSR
27
Δ67-70, P71A,











mut





INTSNETLPR
219
PEIPPNSS
26
IRDSSR
27
Δ67-70, P71A,











mut





INTSNETLPS
25
PKIPPNSS
255
IRDSSR
27
Δ67-70, P71A,





INTSNETLPS
25
PEIPPKSS
256
IRDSSR
27
Δ67-70, P71A,











mut





INTSNETLPS
25
PEIPPNFS
257
IRDSSR
27
Δ67-70, P71A





INTSNETLPS
25
PEIPPNSS
26
IQDSSR
289
Δ67-70, P71A





INTSNETLPS
25
PEIPPNSS
26
IRDSPR
290
Δ67-70, P71A





TPAVKDKLPK
220
PEIPPNSS
26
IRDSSR
27
Δ67-70, P71A,











mut





TNPLNEPLLF
22
PEIPPNSS
26
IQDSSR
291
Δ67-70, P71A,











mut





STHLVPTLRN
221
PEIPPNSS
26
IRDPSR
292
Δ67-70, P71A,











mut





TIPCQSTLNS
222
NDIHLYSS
258
IRDSSR
27
mut










Group N3














TNLSNETLGT
28
PHIRPKVT
30
LIASLR
31

Typical sequence
















TNLSNETLGT
28
PHIRPKVT
30
LIASLR
31






TNLSNETLGT
28
PHIRPKVT
30
LIASLR
31
mut





TDTVLEILRT
223
PHIRPKVT
30
LIASLR
31
mut
61363-C1
2.8
1.8









(G17D)




INTSNETLPS
25
PEIPPNSS
26
LIASLR
31
Δ67-70, P71A,











mut





TNPMNITLHH
224
SQIRPKVT
259
IIASLR
293
mut










Group N4
















TNPLEENLYN
225
YQISTNIP
260
GAHTKD
294
mut
61363-C12
36.0
33.6









(G45E)




TNLSEENLGT
226
PHIRPKVP
261
GAHTKD
295
mut





INHKEGTLPQ
227
GDIHPKFT
262
IVAYLY
296
mut





INTSNETLPS
25
PEIPPNSS
26
INDPLL
297
Δ67-70, P71A





INTSNETLPS
25
PEIPPNSS
26
IIATLR
298
Δ67-70, P71A,











mut





TTPLKERLRK
228
LEISPKSK
263
ISVSLH
299






TNALKDTLII
229
REISPNSS
264
TSYSRG
300
mut





TNPLKETLYN
230
WEIRTNHP
265
SSNSRD
301
mut
61363-H1
22.9
8.4









(V15A)




TIPLQDIL-N
231
FSIRMTPA
266
SFHTHR
302
mut
61362-B8
3.8
2.1









(I23L)




TNTLLETLGT
232
SAISRRSI
267
STESLR
303
mut





TKALKGTLGN
233
WNITQHQT
268
SSDSPR
304






SGTLSGTLLT
234
LDIHPNPT
269
NSNSLW
305






TNTRKDILGT
235
HGIHANTS
270
NSDFLR
306
mut





SNHLKHTLVD
236
RVIRPVST
271
RSVYIH
307






TNAHNTTLLH
237
RAIGQKSE
272
SWHFDR
308






NYPLQETLPK
238
LWIFPNAP
273
SSHSHF
309
mut





TTRPHAALLN
239
SVIQTDPT
274
SYATTR
310
mut





TLPLKESLGT
240
HTIRRTPN
275
SLDTHR
311






ASPMKLTLRY
241
CEIRQYCS
276
RRNPMA
312






TNPLRDTLDT
242
RQIRPIAT
277
WAVAPC
313
mut





TYRHWGTLCK
243
LDIRPDST
278
NTCSLL
314






SSTLSGRLQH
244
QEIRQNST
279
CTYSPW
315
mut





AQPVKLTLRR
245
CEIRLYSM
280
IIGGTL
316






TNRPNEKLRK
246
REIRARST
281
RYDGYC
317
mut





TYTRKEMLHN
247
RDIHENFC
282
RSDNLL
318
mut





TSLVQEPLCH
248
SGIRPYST
283
GRQTLH
319
mut





NCTLKETLIN
249
DEIWTNLI
284
CSNRLL
320






CHTQISTLRY
250
PEIREPCD
285
NSHFLR
321
mut





TRHMTEPLRI
251
RVICIDFI
286
RSYSRR
322
mut





SNGLNGTLRE
252
REILPEST
287
STVSQI
323
mut





Amino acids mutated from wild type after selection are underlined and marked in bold.






Example 12
Measurement of Affinity of Selected NGF ETBPs In Vitro
Selected F13β2 binders were tested for binding to biotinylated β-NGF in radioactive equilibrium binding assay (FIGS. 21A and 21B; Table 4) according to the procedure described for myostatin binders. NGF at concentrations up to 100 nM was incubated with a constant concentration of the purified binder protein (1 or 2 nM) at 30° C. for 1 hour. Affinities to human β-NGF differed greatly between various binders. Proteins from groups N1 and N3 bound NGF only with low affinities: binding to 50 nM of NGF did not exceed 5% of input. Binders from groups N2 and N4 interacted with β-NGF with Kd less than 100 nM. No binding to M-280 beads was observed indicating that the selected molecules interact specifically with human β-NGF.
Example 13
Selection of ETBPs Against Human IgE Receptor FcεRI
The immunoglobulin IgE signaling cascade plays a central role in mediating allergic reactions. Blockade of this pathway has been considered a strategy for treating allergic diseases, including asthma, Crohn's disease, allergic rhinitis and peanut allergy. Upon cross linking by IgE, the high affinity IgE receptor FcεRI initiates the signaling through its cytoplasmic immunoreceptor tyrosine-based activation motif (ITAM). The interaction between IgE and FcεRI involves the two CH3 domains of IgE engaging the extracellular domain of FcεRI (reviewed in Kraft & Kinet, 2007). In principle, the displacement of the interaction partners by competitive molecules should lead to inhibition of this signaling process. The only therapy based on this principle has been the anti IgE antibody (Xolair, Genentech). As another example of ETBP, we have selected binding molecules to the extracellular domain of human FcεRI.
F13β2 mRNA Aext-NNS fusion library was used in in vitro selection against biotinylated human FcεRI. Fusion production and selection procedure was performed similar to myostatin selections. The selection was continued for eight rounds (FIGS. 22A and 22B). To achieve a rapid selection of the binders with high affinities to the target the concentration of FcεRI was dropped twice in each round. Final concentrations of biotinylated human FcεRI used in the selections were as follows: R1 (100 nM), R2 (50 nM), R3 (25 nM), R4 (12.5 nM), R5 (6.25 nM), R6 (3.125 nM), R7 (16 and 1.6 nM), R8 (0.1 nM). Specific binding of selection pool to the target increased after round 5, which is seen in free protein binding profile (FIG. 22B). PCR DNA of PR5 selection pool was cloned into pCR®2.1-TOPO® vector (Invitrogen), and single clones were analyzed by sequencing of 48 clones. The sequences of the binding loops are shown in Table 5. The loop sequences are very different and no sequence motif was selected after round 5 although the protein pool showed specific binding to the target. In the next rounds the pool binding increased significantly (FIG. 22B). The selection was stopped at round 8 following target decrease to 0.1 nM. The binding affinities of selected protein pools after round 7 and 8 were measured in a radioactive binding assay (FIGS. 23A and 23B). The proteins interacted with human FcεRI with very high affinities as low as 5 nM while no non-specific binding to M-280 beads was observed.







TABLE 5







Selected FcεRI binders














SEQ

SEQ

SEQ




ID
DE
ID

ID
Scaffold


BC loop
NO:
loop
NO:
FG loop
NO:
mutation





KGTGSKQ
324
GDIVSS
347
FSAQRPNLVTLL
370
mut


 


QSTMRLW
325
KMILTR
348
YDSGFFPTVVIT
371



 


GRLELRE
326
LTILYP
349
LFVARSKMQLAR
372
mut


 


ELRFPLM
327
ERIMVI
350
TSSKAFDANTNE
373
mut


 


LLVGRAV
328
RLILAR
351
VPGWTGAPMTVN
374
mut


 


GSNKIIL
329
LYIRIN
352
AATKRLRYKPAA
375
mut


 


EDKVTHS
330
TFIFXF
353
SCPDAVXTKSTK
376



 


LGHEVAE
331
MVIQSV
354
LKLLLDSSVGST
377
mut


 


LSNASMP
332
TCIWXC
355
LRLSGWRIFNTK
378
mut


 


AAKITMW
333
LVIVPR
356
NVNRVEPMGLPP
379
mut


 


GEYKDPT
334
VRIWFP
357
LRSMKIWTNDTS
380
mut


 


MVPEGAS
335
NSIERL
358
GHDESPKNRSAD
381



 


DYMARAQ
336
QSISQG
359
ANADLIIHGTNL
382
mut


 


CSTVKIR
337
TQIDRP
360
TKRWNTADLSAR
383



 


PRWEPGE
338
YIIFRI
361
VDGADVLWTLRP
384



 


APKLKDD
339
RIIFPW
362
LLFARSNQGWKV
385



 


RPPRRLL
340
GKITLQ
363
FLLWVHSFVSRW
386
mut


 


PRPIQII
341
GFIQAI
364
KDPYHCKPHDVS
387
mut


 


LLTTSFR
342
NMILLP
365
LPVIIKTNSNQV
388
mut


 


QEILSNL
343
NVIVMI
366
ISTVANVDTGTR
389



 


RPDEDGS
344
EMIMER
367
LSWTKVDKTYVV
390
mut


 


VQHYLAR
345
FGIDDP
368
IYGPDLEVCRLW
391



 


LMAGDSR
346
TLIAPY
369
LSDGGIRGQWMV
392
mut





Amino acids mutated from wild type after selection are underlined and marked in bold.






Example 14
Characterization of Wild Type F13β1 Scaffold
According to the crystal structure two distinct domains with a seven-stranded β-barrel fold are located at the carboxyl terminus of the FXIII-A monomer: β-barrel 1 and β-barrel. Domain β-barrel 1 contains residues S517 through L628 and is a little larger than β-barrel 2 due to its loop segments. The two barrels are structurally very similar, but there is no significant sequence similarity between them. Both of these domains were shown to be highly thermostable. To characterize the expression of an isolated barrel 1 domain in E. coli we amplified F13β1 sequence (S517-L628) from full length factor XIIIA cDNA clone (SC120099; OriGene Technologies, Inc., Rockville, Md.) by PCR with primers oligo 60 and oligo 62rev to introduce NdeI at 5′ end, and His6-tag-Stop sequence (“His6” disclosed as SEQ ID NO: 29) and Barn HI site at 3′ end, respectively. The resulting fragment was cloned into NdeI and BamHI sites of a modified pet28(a+) vector. The map of the plasmid (modified pet28(a+)-F13(1-His6 “His6” disclosed as SEQ ID NO: 29)) is shown in FIG. 24A. Tagless F13β1 domain and F13β1-GFP fusion were constructed by using primers oligo EG1rev and oligo 61rev for 3′ end in PCR, respectively. The correct sequence of F13β1 scaffold was confirmed by sequencing. The sequence corresponded to the published sequence of Factor XIII-A (accession number NP_000120) (FIG. 25). The protein was expressed in E. coli BL21 (DE3) pLysS cells (Invitrogen) under different temperature conditions, and the solubility profile was assessed as described above for F13β2 myostatin binders. Although a small portion of F13β1 protein was soluble at 18° C. (FIG. 26A) and could be purified via affinity chromatography, the majority of the protein remained in the insoluble fraction.
To characterize the ability of an isolated barrel 1 domain to autonomously fold in E. coli a fusion of the human β-barrel 1 domain joined at its C-terminus to GFP was studied for solubility in 1×PBS, pH 7.4. Although F13β1-GFP protein remained in insoluble pellet it was correctly folded, which was indicated by strong green fluorescence of GFP in the pellet. Previously, it was shown that both β-barrel domains of FXIII-A remained folded at low pH. Using the acidic condition (36 mM HCl) applied earlier for purification of F13β2 myostatin binders we were able to dissolve the F13β1 scaffold (FIG. 26A). Thus high solubility in low pH is a feature of not only isolated domains but also of proteins with more than one barrel of the Transglut_C family. As the behavior of the fusion with GFP indicates, fusions to transglutaminase barrel proteins can be used to allow the fused moiety to be purified by selective solubility at low pH.
Example 15
Diversification of F13β1 Scaffold
The β-barrel 1 domain of Factor XIII is also a good candidate for an alternative scaffold either in the form of a single domain or as an element of a F13β1β2 heterodimer (FIGS. 27A and 27B). Similar to the F13β2 scaffold six potential loops for randomization are present in F13β1 structure: at least 21 and 33 residues can be mutated in the N-terminal loop region and the C-terminal loop region of the domain, respectively (FIG. 28).
By utilizing F13β1β2 heterodimer as a scaffold several binding sites could be placed on one molecule: for example, six variable loops would contribute to the binding site via randomization of F13β1 C-terminal loop region and F13β2 N-terminal loop region (FIG. 27B).
Example 16
Characterization of Wild Type F13β1β2 Heterodimer
To construct a F13β1β2 heterodimer wild type optimized F13β2 domain was amplified by PCR with oligo 129 and oligo 126rev to introduce NdeI site and part of F13β1 domain (positions D617-L627) at 5′ end, and PSM-His6-tag-Stop sequence (“His6” disclosed as SEQ ID NO: 29) and Barn HI site at 3′ end, respectively. To insert the additional cloning site AatII two silent mutations were introduced at D618 and V619 positions of F13β1 domain. The resulting fragment was cloned into NdeI and BamHI sites of a modified pet28(a+) vector. F13β1 domain (S517-L628) was amplified from full length factor XIIIA cDNA clone (SC120099; OriGene Technologies, Inc., Rockville, Md.) by PCR with primers oligo β2 (NdeI) and oligo β6 rev (AatII) and cloned into NdeI and AatII sites of modified pet28(a+) vector, containing F13β2 domain (see above). The map of the plasmid (modified pet28(a+)-F13β1β2-His6 “His6” disclosed as SEQ ID NO: 29)) is shown in FIG. 24B. To generate proteins, containing PSM-Stop sequence or GFP fusion at the C-terminus, primers oligo AKx299 (XhoI) or oligo 52rev (Bam HI) were used for 3′ end in PCR, respectively. The protein was expressed in E. coli BL21 (DE3) pLysS cells (Invitrogen) under different temperature conditions, and solubility profiles were assessed as described above for F13β2 myostatin binders. A small fraction of F13β1β2 heterodimer was soluble at 18° C. (FIG. 26B) and could be purified via affinity chromatography. To characterize the folding of an isolated β1β2 heterodimer in E. coli an F13β1β2-GFP fusion was studied for solubility in 1×PBS, pH 7.4. Correct folding was suggested by the strong GFP signal of the insoluble pellet. As for the F13β1 domain, the heterodimer of β1 and β2 barrels could be solubilized by exposure to acid (FIG. 26B).
Example 17
Display of ETBPs on Bacterial Phage
Phage display has been widely used in selecting peptides and proteins that bind desired targets with high affinity and selectivity. A translational fusion is made between the displayed peptide or protein and a phage coat protein. When the fusion protein is expressed and assembled into a phage particle in bacterial host cells, a physical linkage of phenotype (displayed protein or peptide) and genotype (DNA encoding the displayed protein or peptide) is made, which renders reiterated selections feasible. In the current invention, we have developed reagents and methods for the display of discrete or sequence-diversified ETBPs in phagemid vectors based on the filamentous bacterial phage M13. We describe construction of phage display vectors and diversified libraries of FXIIIβ barrel proteins. We demonstrate their untility for identifying ETBPs by affinity selection using phage display.
Display Vector Design
Filamentous bacterial phage M13 was employed to display ETBPs. Both phage and phagemid vectors have been used in phage display in general. Phage vectors typically encode all phage proteins as well as the displayed peptide or protein fused to a phage coat protein. A phagemid is a plasmid vector that contains an E. coli origin of replication and a phage origin for single stranded DNA production to allow its packaging into phage particles when other phage proteins are expressed from a helper phage genome. A number of phage coat proteins have been used as fusion partners and the product of gene III (pIII) has been the most widely used. In the absence of wild type pIII expression, five copies of fusions are expected to be displayed on a phage particle. When wild type pIII is expressed from the helper phage genome, pIII fusion copy numbers are variant depending on the display system, theoretically ranging from 0-5 copies per phage. Display copy number is an important parameter that can alter the outcome of selection. When the copy number is more than one, an avidity effect allows display phage to bind targets at an apparent affinity better than the displayed molecule's intrinsic affinity when it is uncoupled from the phage. Multivalency exerts in an avidity effect on targeting binding which renders inefficient discrimination of binders with different affinities in selections, but can be advantageous when low affinity binders are expected from a library such as short peptides. The ability to modulate display copy number is therefore preferred in a phage display system. This can be achieved through a number of strategies, including different fusion partner (pIII or pVIII, for example), different helper phage and the use of promoters that are amenable to regulation. We have employed the lacZ and pBAD promoters to display ETBPs to provide different ways to modulate fusion copy number. The pBAD promoter is known for its tight regulation. AraC positively regulates pBAD and also acts as a repressor of its own expression. pBAD has been proposed for phage display of proteins that might be toxic for the host cells. The lacZ promoter has been widely used in phage display. Basal expression from lacZ promoter is commonly used in displayed fusion protein expression. The possibility to use inducer or repressor to modulate expression makes the lacZ promoter an attractive choice for phage display. In the current invention, we provide methods and compositions using phagemid vectors to display ETBPs.
Construction of Phagemids FXIIIβ2pIII, FXIIIβ2pIIICT, E3pIII, E3pIIICT, FXIIIβ2BbsIpIIICT and E3BbsIpIIICT
We have constructed a number of phagemid vectors for efficient cloning of highly diverse ETBP library DNA sequences and for functional display of discrete ETBPs as pIII fusions. FIG. 29 outlines key features of six phagemids. The myostatin binding ETBP E3 as well as the wild type FXIII barrel 2 domain (FXIIIβ2) were fused in frame to either the mature full length pIII or the C-terminal domain of pIII. FXIIIβ2pIII, FXIIIβ2pIIICT, E3pIII, and E3pIIICT were constructed by an overlapping PCR strategy where FXIIIβ2 and E3 fragments were made by PCR using primers FG2 and FG3 and their cognate plasmid DNA as template. DNA fragment encoding the mature full length pIII protein was made using primers FG4 and FG5 and helper phage M13K07 (New England Biolabs, Beverly, Mass.) as template. DNA fragment encoding the C-terminal domain of pIII was similarly made using primers FG6 and FG5. PCR reactions were carried out on a DNA engine Tetrad (MJ Research): 94° C., 3 minutes followed by 25 cycles of 94° C., 30 seconds; 55° C., 30 seconds and 72° C., 2 minutes. A further 5-minute extension was added after the last cycle. The 50 μl reactions included 200 μM dNTPs, 0.4 μM each primer, approximately 4 ng template plasmid, 5 units Herculase® Hotstart DNA Polymerase and reaction buffer (Stratagene, La Jolla). The fusion DNA fragments were assembled by annealing and extending the overlapping PCR fragments, resulting in coding sequences for translational fusions of FXIIIβ2 or myostatin binding ETBP E3 to mature full length or C-terminal domain of pIII. Overlapping PCR fragments were separated by agarose gel electrophorosis and purified using the Qiaquick Gel Extraction kit (Qiagen). Two corresponding purified overlapping DNA fragments (5μ each) were mixed with 200 uM dNTPs, 2 units Herculase® Hotstart DNA Polymerase and reaction buffer (Stratagene, La Jolla) in a total volume of 15 pl. Annealing and extension were carried out on a DNA engine Tetrad (MJ Research): 94° C., 3 minutes followed by 10 cycles of 94° C., 30 seconds; 55° C., 30 seconds and 72° C., 2 minutes. A further 5-minute extension was added after the last cycle. One ul of the assembled DNA fragments was amplified in a 50 μl reaction by PCR with primers FG1 and FG5. PCR reactions were carried out on a DNA engine Tetrad (MJ Research): 94° C., 3 minutes followed by 25 cycles of 94° C., 30 seconds; 55° C., 30 seconds and 72° C., 2 minutes. A further 5-minute extension was added after the last cycle. The 50 μl reactions included 200 uM dNTPs, 0.4 μM each primer, 5 units Herculase® Hotstart DNA Polymerase and reaction buffer (Stratagene, La Jolla). The resulting PCR products were gel-purified, digested with EcoR I and Hind III, and cloned into Hind III and EcoR I digested puc119 (Maxim Biotech, South San Francisco). FXIIIβ2BbsIpIlICT (sequence set forth in Table 15, FIGS. 30A and 30B) and E3BbsIpIlICT were constructed by a modification of E3pIIICT to include two Bbs I sites flanking FXIIIβ2 or E3. The FXIIIβ2 and E3 DNA fragments were generated by PCR using primers AKx295Y and AKx297Y with their cognate plasmid template. PCR reactions were carried out on a DNA engine Tetrad (MJ Research): 94° C., 3 minutes followed by 25 cycles of 94° C., 30 seconds; 55° C., 30 seconds and 72° C., 2 minutes. A further 5-minute extension was added after the last cycle. The 50 ul reactions included 200 uM dNTPs, 0.4 uM each primer, approximately 4 ng template plasmid, 5 units Herculase Hotstart DNA Polymerase and reaction buffer (Stratagene, La Jolla). The PCR products were purified with Qiaquick PCR purification kit (Qiagen), digested with Bgl I and Not, gel-purified with Qiaquick Gel Extraction kid (Qiagen) and cloned into Sfi I and Not I digested E3pIIICT. DNA sequences of the oligonucleotide primers used in construction are shown in Table 10. Successful constructions were confirmed by DNA sequencing of resulting bacterial clones. The resulting phagemids encode the translational fusions between FXIIIβ2 or E3 to pIII or pIIICT with a 6-his (SEQ ID NO: 29) and a myc tag for purification and detection. Two Mlu I sites were included for excision of the pIII coding sequence when expression of ETBPs as non-fusion is desired. Maps of phagemids FXIIIβ2BbsIpIIICT and paracompFXIIIβ2 are provided in FIG. 30. The complete sequence of the FXIIIβ2BbsIpIlICT phagemid is provided in Table 12.
Construction of paracompgp3 and paracompFXIIIb2pIII
The sequence of the paracompFXIIIb2pIII plasmid was generated by replacing the stuffer fragment of a phagemid vector paracompgp3 that had been constructed in three steps: (i) taking a NotI to Bsu36I fragment from pacycaracvce as the vector fragment and as the insert fragment, a NotI+Bsu361 digested PCR fragment of the M13 pIII gene using primers (#1867 & #1868 to amplify the pIII gene from M13 phage DNA); (II) inserting the phage M13 origin of replication into the above plasmid by a three part ligation consisting of: a vector fragment created by BsmBI+HindIII digestion of a PCR fragment created by amplification of the contiguous SV40 ori, pBR322 origin, beta lactamase and rrnG terminator segments of pEAK15Flpe (#1875/#1876 as primer and peak15flpe as template); Insert 1, PCR BsmB1/bsu36 M13 on fragment (#1873/#1874 as primers); and Insert 2, plasmid made from step 1, cut with HindIII and Bsu361; and finally (iii) replacing the existing insert sequences between PstI and NotI with a short synthetic DNA segment prepared from kinased oligos#1882/#1883. The complete sequence of the paracompgp3 phagemid is provided Table 13.
Functional Display of Discrete ETBPs
Functionality of displayed E3 was confirmed by E3pIII and E3pIIICT phage binding to myostatin, as shown in FIG. 31. Both E3pIII and E3pIIICT phage bound immobilized myostatin in Elisa, while FXIIIβ2pIII and FXIIIβ2pIIICT phage showed little binding (FIGS. 31A and 31B). Phage infectivity was retained after target binding, whether phage was eluted with a low pH solution or remained bound on Elisa vessel (direct infection, FIG. 31B), indicating robustness of the ETBP displaying phage. 100-1000 more phage were recovered from E3 phage than FXIIIβ2 phage, indicating efficient discrimination against the non-binding FXIIIβ2 phage. E3pIII and E3pIIICT phage binding to target was highly selective as they bound two myostatin preparations (myostatin and biotinylated myostatin) while little binding was detected when control proteins mbp-FcεRI and BSA were used in place of myostatin (FIGS. 32A and 32B). Myostatin binding Elisa was performed as described below. Wells of Maxisorb plate (Nunc) were incubated with 100μ/well with 2 μg/ml myostatin or control proteins in a bicarbonate coating buffer, pH 9.6 at room temperature for 1 hr. Wells were washed and blocked with BSA. Phage dilutions were incubated with coated and blocked wells at room temperature for 1 hr. After washes, bound phage was detected with an anti-M13 monoclonal antibody HRP conjugate (GE).
Under basal expression conditions, E3pIII and E3pIIICT phage bound myostatin strongly. When high affinity binders are desired from a selection, a display copy number of no more than one is preferred. Glucose is known to decrease expression from the lacZ promoter by lowering intracellular cAMP. As shown in FIGS. 33A and 33B, phage binding to myostatin is diminished when it is propagated in culture media supplemented with increasing glucose concentrations. The dynamic range of this regulation appears broad for practical implementation in selection. For example, when binding reached near saturation with no added glucose (0%), little binding was detected with highest added glucose (2%) when phage input was similar in the binding reactions.
E3pIII and E3pIIICT protein levels also decreased in phage produced in media containing increasing concentrations of glucose, as indicated in phage Western shown in FIG. 34. This decrease is likely due to a combination of reduced copy number per phage and the number of phage containing at least one fusion copy. Phage Western was performed as described below. Phage from 500 μl culture supernatant was prepared by PEG-8000 and NaCl precipitation. Phage pellet was dissolve in 100 μl PBS pH 7.4 and 10 μl was analyzed on a 15% SDS PAGE and eletrotransferred to a PVDF membrane, which was first probed with an anti-pIII antibody (MoTec) followed by a goat anti-mouse Fc-HRP.
FXIIIβ2 and E3 were also placed under the control of the arabinose inducible pBAD promoter in the phagemids paracompFXIIIβ2pIII and paracompE3pIII, respectively. FIG. 35 shows that phage produced from paracompE3pIII bound myostatin in Elisa. When paracompE3pIII phage was produced in growth media supplemented with arabinose, its binding to myostatin increased in an arabinose concentration dependent manner. Little binding to myostatin was detected from paracompFXIIIβ2pIII phage.
Construction of Diversified ETBP Libraries
An ETBP phage display library with a sequence diversity of 5×108 was constructed based on the strategy outlined in FIG. 36. It is based on the A-50% NNSFGext library randomization strategy in which 9 positions in BC loop (50% NNS, 50% WT), 7 positions in DE loop (50% NNS, 50% WT), and 12 positions in FG loop (NNS) were randomized. Approximately 2 pmols of the A-50% NNS FGext diversifed DNA was amplified by PCR using oligonucleotide primers AKx296Y and AKx298Y in a total volume of 1 ml. PCR reactions were carried out on a DNA engine Tetrad (MJ Research): 94° C., 3 minutes followed by 25 cycles of 94° C., 30 seconds; 55° C., 30 seconds and 72° C., 2 minutes. A further 5-minute extension was added after the last cycle. The 1 ml PCR reaction included 200 uM dNTPs, 0.4 uM each primer, 100 units Herculase® Hotstart DNA Polymerase and reaction buffer (Stratagene, La Jolla). The amplified DNA was purified using Qiaquick PCR purification kit (Qiagen) and digested with Bbs I. The digested DNA was purified again using Qiaquick PCR purification kit (Qiagen). Five μg of this DNA was ligated to 20 μg of Bbs I digested E3pIIICT phagemid DNA in 2 mls using T4 DNA ligase (New England Biolabs). The reaction mixture was incubated at 16° C. overnight and precipitated with glycogen (Ambion) and ethanol. The resulting DNA dissolved in water and used to transform E. coli TG1 cells in 22 separate eletroporations using a Gene Pulser (Bio-Rad).
One ml of SOC media was added to cells after each electroporation. The electroporated cells were pooled (approximately 24 mls) and grew in 37° C. in a 250 ml flask with 250 rpm shaking for 40 minutes. Transformation efficiency was determined by plating serial dilutions of this culture on LB plates containing 50 μg/ml ampicilin and 1% glucose. Total number of transformants was calculated to be 5×108.
The transformed cells (24 m) were transferred to 1 L 2×YT in a 3 L baffled flask containing 50 μg/ml carbenecilin and grew in 37° C. with 250 rpm shaking for 2 hr. Cells from an aliquot of this culture (200 ml) culture were collected by centrifugation. The cells were resuspended in 2×YT. An equal volume of 30% glycerol was added to the resuspended cells and stored in −80° C. The helper phage M13K07 (amplified from New England labs) was added to the remaining culture (800 ml) to a final titer of 1×10e10 pfu/ml to produce display phage by incubation at 37° C. overnight with 250 rpm shaking. Library phage was harvested by centrifugation and precipitated by adding 1/5 volume of 20% PEG-8000 and 2.5 M NaCl. Precipitated phage was purified by centrifugation and resuspended in PBS pH 7.4, adjusted to 50% glycerol and stored in −20° C. Phage library titer was determined to be 1×10e13 cfu/ml by infecting E. coli TG1 cells and counting ampcilin resistant colony forming units. DNA sequencing of resulting library members (96 clones from electroporation and 96 clones from infection by library phage) revealed conformity to design. No duplicates of diversified sequences were observed in the sequenced clones. A summary of library sequencing analysis is shown in Table 6.
Selections against several protein targets were carried out to validate display of sequence-diversified β barrel proteins. Library phage (200 ul, 5×10e11 cfu/ml) was added to a Maxisorp well coated with target protein and incubated at room temperature for 1 hr. Liquid was discarded and the wells were washed 10 times with 200 Elisa wash buffer (50 mM Tris-HCl, pH 8.0, 138 mM NaCl, 2.7 mM KCl, 0.05% TWEEN-20TM). Bound phage was eluted with 200 μl 0.2 M glycine, 0.1 M HCl, pH 2.2 by incubating at room temperature for 10 min. Eluent was transferred to tube containing 30 μl 1M tris-HCl, pH 9.1. The eluted phage was amplified by mixing 100 μl of elution with 2 ml mid log phase TG1 cell (A600=0.7) and growing at 37° C. for 1 hr. This culture was mixed with 25 ml 2YT containing M13K07 (1×1010 pfu/ml) and carbenecillin (50 μg/ml) in a 250 ml flask and grown at 37° C. overnight with shaking at 250 rpm. Amplified phage was purified by PEG precipitation and used for next round of selection.
Table 8 shows a summary of DNA sequencing analysis of randomly picked clones after each round of selection. Fast sequece convergence was observed in the myostatin selection: 80%, 100% and 100% of sequenced clones share identical sequence after round 1, 2 and 3, respectively. The abundance ( ̃1% in library by sequencing analysis) and affinity of the winning clone were likely the main reason for the observed fast enrichment. This result, however, demonstrates the possibility of functional display of ETBPs on the M13 phage. The selection process also efficiently eliminated non-displayable clones, such as those with stops codons and frameshifts. This is evident in the sequencing summary shown in Table 6.







TABLE 6







Sequencing analysis of phage display library (A50% FG-extNNS)












number
in frame
del &
in frame



analyzed
full length
frame shift
stop















5x10e8 library ligation
83
18
32
33


5x10e8 library phage
91
14
39
38


FcεRI PR1
16
11
3
2


FcεRI PR2
15
13
1
1


FcεRI PR3
16
11
4
1


FcγRIIb PR1
16
6
6
4


FcγRIIb PR2
15
10
3
2


FcγRIIb PR3
14
6
5
3


CD5-FC PR1
15
5
9
1


CD5-FC PR2
16
10
2
3


CD5-FC PR3
16
11
1
4


CD19D2D3-FC PR1
16
11
3
2


CD19D2D3-FC PR2
13
11
1
1


CD19D2D3-FC PR3
16
9
5
2


Myostatin PR1
16
15
1
0


Myostatin PR2
16
16
0
0


Myostatin PR3
16
16
0
0


ActRIIb PR1
15
10
3
2


ActRIIb PR2
13
13
0
0


ActRIIb PR3
15
9
4
2









Phagemid Modifications

A number of modifications were made to the phagemid vector FXIIIβ2BbsIpIIICT, including reversing the M13 (+) origin of replication to M13 (−), creating restriction sites through silent mutations to facilitate loop shuffling, and introducing stop codons in each of BC, DE and FG loops. These modifications allow construction of highly diversed libraries by increased transformation efficiency over commonly used restriction fragment ligation.
The DNA sequence of the resulting phagemid pF13 wt-modstoppIIICT is shown in Table 16.
Construction of Phage Display Libraries with Higher Diversity
Two phage display libraries, FG6 and FG6+6, each with a sequence diversity of approximately 1×1010, were constructed using the FXIII β barrel 2 scaffold. Targeted randomization of the BC, DE and FG loops were achieved by oligo-directed mutagenesis. The two libraries share the same BC and DE loop randomizations strategy, but differ in their FG loop randomizations. Six amino acid positions were randomized for the FG 6 library and an additional 6 randomized amino acids were inserted into the FG loop for the FG 6+6 library. FIG. 37 outlines the randomization strategy. A mixture of F13BC, F13DE plus F13FG6 or FG6+6 were phosphorylated in a reaction by T4 polynucleotide kinase. At 3 molar excess, the kinased oligos were annealed to pF13 wt-modstoppIIICT single stranded DNA containing uracil after incubating at 90° C. for 3 min, 50° C. for 5 min and 20° C. for 5 min. Following annealing, T7 DNA polymerase and T4 DNA ligase were added to complete the enzymatic synthesis of double-stranded DNA, which was then purified using the QIAquick PCR Purification Kit (Qiagen). Twenty μg of the purified DNA was used in a single electroporation of 350 μl of LC1062 E. coli competent cells. 1×1010 transformants were obtained. Library phage was produced and purified by polyethlene glycol precipitation using standard protocols. DNA sequencing analysis of randomly picked clones of library members reveal that greater than 90% of clones had mutations in at least one of the targeted loops, including approximately 40% of clones had mutations in all three targeted loops. Twenty percent of the library members had mutaions in all three loops that did not include a stop codon. Table 7 shows a summary of the FG6 and FG6+6 library sequence analysis.







TABLE 7







Sequencing Analysis of FG6 and FG6+6 Libraries











Randomization Outcome
FG6, n = 80
FG6+6, n = 77















None
5
5



BC
2
1



DE
2
0



FG
0
2



BC, DE
7
2



DE, FG
1
1



BC, FG
4
3



BC, DE, FG
32
34



BC, DE, FG no stop codon
15
17



Frameshift
12
12










Selection from FG6 and FG6+6 Libraries

Affinity selections to immobilized FcgRI-MBP and lysozyme were carried out from both FG6 and FG6+6 libraries. Library phage (1013 pfu/ml in PBS pH 7.4, 0.5% w/v BSA, 0.05% TWEEN-20TM) was incubated for 1 hour at room temperature with Maxisorp wells coated with FcεRI-mbp (produced in E. coli) and chicken lysozyme (Sigma). After removal of unbound phage and washes with PBS pH 7.4, 0.05% Tween-20, bound phage was eluted with 100 mM HCl and neutralized with 1/8 volume of 1 M Tris, pH 11. Eluted phage was titered and amplified in LC1062 cells for subsequent round of selection. Four rounds of selections were carried out using similar conditions. Phage titer from control wells without target coating was also determined to assess enrichment. FIG. 38 shows phage titers of the four selections. A general trend of increased titer was observed as selection progressed with modest specific enrichment (target vs. control) after the forth round of selection.
An Elisa screening was performed after four rounds of selection to identify binding clones. Randomly picked phagemid clones were incubated with target-coated or buffer-coated Maxisorp wells at room temperature for 1 hour. Unbound phage was removed and the wells were washed eight times with PBS pH 7.4, 0.05% Tween-20. Binding was determined with an anti-M13 pVIII antibody-HRP conjugate (GE). FIG. 39 shows that some selected clones exhibited highly specific binding to their cognate targets. Varying levels of binding to targets and the Maxisorp surface were also observed in other clones. DNA sequencing analysis indicated clear enrichment of certain sequences, as shown in Table 8.







TABLE 8







Sequencing analysis of selected clones.


Table 8 discloses “BC loop” sequences as SEQ ID NOS 393-403, the 


“DE loop” sequences as SEQ ID NOS 404-414 and the “FG loop” sequences


as SEQ ID NOS 415-425, all respectively, in order of appearance.
















DE




Sequencing
Target
Well


BC loop
loop
FG loop
n
Library
Target
plate ID
Binding
Binding





TNPLKET
RPNST
SDSLRH
n.a.
n.a.
n.a.
n.a.
n.a
n.a


 


MNPRMEM
HPDSD
RLWTLWMARWRW
10/26
FG6 + 6
FcεRI-MBP
81937
2.35
1.31


 


SNPLSRP
RPHSN
SRSLWLSIGKRH
 9/26
FG6 + 6
FcεRI-MBP
81937
3.31
2.08


 


TNPLRGT
PPNVK
GLSTAGRWKRVR
 4/26
FG6 + 6
FcεRI-MBP
81937
4.00
1.10


 


GNPTKET
RPRSM
YRPRRGGMRLRH
 2/26
FG6 + 6
FcεRI-MBP
81937
4.00
1.47


 


TNPRRRV
RPRST
WNSRWPAPTRRR
 1/26
FG6 + 6
FcεRI-MBP
81937
1.73
0.31


 


INPLQVS
GPSSA
IYRYGL
22/28
FG6
lysozyme
81938
4.00
0.10


 


ANPIKHM
RPTAG
LGVLRR
 2/28
FG6
lysozyme
81938
2.83
1.76


 


RRPTKGN
GPGQV
LGMLRR
 2/28
FG6
lysozyme
81938
2.45
1.33


 


TIPHRGR
QPNVP
RAFLRH
 1/28
FG6
lysozyme
81938
0.34
0.25


 


PSPFIEN
RPECI
RGWLRS
 1/28
FG6
lysozyme
81938
1.19
0.80









Example 18
Pharmacokinetic Characterization of Pegylated ETBPs
Conjugation of drugs to polyethylene glycol (PEG) has been shown to improve pharmacokinetic property and reduce immunogenicity of parent drug molecules. Such modifications have generally resulted in prolonged half life from minutes to hours and from hours to days. Pharmacokinetic studies were carried out to measure the in vivo half life of PEGylated ETPBs. Subject animals (FBV) were injected subcutaneously (s.c.) at 5 mg/kg PEGylated myostatin binder F10 (monomer)-PEG or B8 (tandem F10 dimer). Blood samples were collected at 0, 1, 2, 4, 8, 24, 32, 48, 56, 72 hr. after injection and placed into tubes containing 1.5 ul 0.5 M EDTA. Plasma samples were collected by centrifugation at 6000 rpm for 10 min. The F10 and B8 concentrations in plasma was determined by Elisa using an anti-PEG antibody (Epitomics) as a capture antibody and followed by biotinylated myostatin and streptavidin-HRP (Zymed) for detection. Quantitation was determined by a non-linear regression (Graphpad Prism) to F10 and B8 standards. Pharmacokinetic modeling was carried out using linear regression of log concentration (y) vs time (x) based on a non-compartmental extravascular model (200, WinNonlin). FIGS. 40A and 40B shows two examples of such modeling results. The summary in Table 9 indicated that F10 and B8 had an average in vivo half life of 25 hr. and 16 hr., respectively. These PK profiles are similar to those of a number of PEGylated drugs. F10 appears to have a better PK profile than B8: a longer half life and better bio-availability (average Cmax 22.25 vs. 5.9 ug/ml). The functionality of F10 and B8, as measured by their binding to myostatin in Elisa, did not appear to have changed during the PK study period, suggesting a favorable in vivo stability profile for these proteins.







TABLE 9





In viov Pharmacokinetic Properties of PEGylated ETBP F10 and B8






















F10 #1
F10 #2
F10 #3
F10 #4
F10 #5
F10 #6





HL-Lambda z 
18.3
17.1
27.3
26
29.2
31.2


(hr)


Rsq
0.98
0.94
0.97
0.98
0.99
0.97


Cmax, ug/ml
24.8
21.2
20.4
21.7
20.3
25.1


Tmax, hr
8
24
24
8
24
24






B8 #1
B8 #2
B8 #3
B8 #4
B8 #5
B8 #6





HL-Lambda z
12.1
8.7
6.7
16.7
25.8
25.6


(hr)


Rsq
0.99
0.99
0.99
0.96
0.95
0.96


Cmax, ug/ml
5.9
7.2
6.4
8.5
3.1
4.3


Tmax, hr
8
4
4
8
2
1
















TABLE 10







Primers used for cloning and construction.


Table 10 discloses “His6” as SEQ ID NO: 29 and


“G4S” as SEQ ID NO: 32.











SEQ




ID


Primer
Sequence
NO:










1. Primers for wild type F13β2 scaffold.









oligo 17
TAATACGACTCACTATAGGGACAATTACTATT
426


(NdeI)
TACAATTACATATGACCATCCCTGAGATCATC




ATCAAG



 


oligo 18rev
TTTTTTTTTTTTTTTTTTTTAAATAGCGGATG
427


(BamHI)
CCTTGTCGTCGTCGTCCTTGTAGTCGGATCCT




CGTCTTTGAATCTGCACGTCCAG



 







2. Primers for optimized F13β2 scaffold.









oligo 53
GAAATAATTTTGTTTAACTTTAAGAAGGAGAT
428


(NdeI)
ATACATATGACCATCCCTGAGATCATCATCAA




G



 


oligo 19
ATGACCATCCCTGAGATCATCATCAAGGTCCG
429


(or 67I)
CGGCACTCAGGTCGTGGGTTCTGACATGACTG




TGATCGTGGAGTTT



 


oligo 20rev
CAGGTGCACCCAGACATTGCGCAGGGTTTCTT
430


(80revI)
TCAGAGGATTGGTAAACTCCACGATCACAGTC




AT



 


oligo 21
CTGCGCAATGTCTGGGTGCACCTGGATGGTCC
431



TGGAGTCACACGCCCAATGAAGAAGATGTTCC




GCGAAATC



 


oligo 22rev
CACTTCTTCCCACTGCACGGTGGAGTTTGGGC
432


(82revI)
GGATTTCGCGGAACATCTTCTTCAT



 


oligo 23
GTGCAGTGGGAAGAAGTGTGCCGCCCATGGGT
433



CTCTGGGCATCGCAAGCTGATCGCCAGCATG



 


oligo 25rev
GCGGCGTTGAATCTGCACGTCCAGCTCGCCAT
434



ACACATGGCGCAGGGAGTCACTGCTCATGCTG




GCGATCAGCTT



 


oligo 28rev
TAGATCATTGGATCCCTCATTAATGATGGTGA
435


(BamHI-His6)
TGGTGATGGCGGCGTTGAATCTGCAC



 


oligo 52rev
TCCTTTGCTGAATTCGCCAGAACCAGCAGCGG
436


(BamHI)
AGCCAGCGGATCCGCGGCGTTGAGCAC



 


oligo 48rev
GACATTGCGCAGGGTTTCTTTCAGAGGATTGG
437



TAAACTCCACGATCACAGTCAT



 


oligo AKx198
TAGATTATTCTCGAGCTCATTAATGATGGTGA
438


(PSM-His6)
TGGTGATGCATGGACGGGCGGCGTTGAATCTG




CAC



 


oligo AKx299
TAGATTATTCTCGAGCTCATTACATGGACGGG
439


(PSM)
CGGCGTTGAATCTGCAC



 


oligo AKx226
TAGATTATTCTCGAGCTCATTACATGCACGGG
440


(PCM)
CGGCGTTGAATCTGCAC



 


oligo AKx169
TAGATTATTCTCGAGCTCATTAATGATGGTGA
441



TGGTGATGGCGGCGTTGAATCTGCAC



 


AKx202
TATTATTCTAGAAATAATTTTGTTTAACTTTA
442



AGAAGGAGATATACCATGGGGACCATCCCTGA




GATCATCAT



 


AKx203
AGTAGTAGTCATATGSNNSNNSNNSNNSNNGC
443



GGCGTTGAATCTGCAC



 







3. Primers for site-directed mutagenesis.









oligo 51
GTGCAGTGGGAAGAAGTGNNSCGCCCATGGGT
444


(C69X)
CTCTGGGCATCGCAAGCTGATCGCCAGCATG



 


oligo 49
AAAGAAACCCTGCGCAATGTCNNSGTGCACCT
445


(W38X)
GGATGGTCCTGGA



 


oligo 50rev
GCGGCGTTGAATCTGCACGTCCAGCTCGCCSN
446


(Y92X)
NCACATGGCGCAGGGAGTC



 







4. Primers for F13β2 libraries construction.









oligo 63 (or
GTGCAGTGGGAAGAAGTGGTGCGCCCATGGGT
447


71I) (C69V)
CTCTGGGCATCGCAAGCTGATCGCCAGCATG



 


oligo 39
GTCTGGGTGCACCTGGATGGTCCTGGAGTCAC
448



ACGCCCAATGAAGAAGATGTTC



 


oligo 55
TAATACGACTCACTATAGGGACAATTACTATT
449


(T7Tmv)
TACAATTACAATGACCATCCCTGAGATCATC



(or 78I)




 


oligo 26 rev
TTTTTTTTTTTTTTTTTTTTAAATAGCGGATG
450


(Flag-tag)
CCTTGTCGTCGTCGTCCTTGTAGTCGCGGCGT



(or 74revI)
TGAATCTGCACGTC



 


oligo 65rev
GTCGTCGTCCTTGTAGTCGCGGCGTTGAATCT
451


(RT primer)
GCACGTC



(or 76revI)




 







N-terminal loop libraries









oligo 35 rev
CAGGTGCACCCAGACATTGCGCAGSNNSNNSN
452


(BC C-NNS)
NSNNSNNATTGGTAAACTCCACGATCACAGTC




AT



 


oligo 41rev
CACTTCTTCCCACTGCACGGTSNNSNNSNNSN
453


(DE C-NNS)
NGATTTCGCGGAACATCTTCTTCATTGGGCG



 


oligo 45rev
GCGGCGTTGAATCTGCACGTCCAGCTCGCCAT
454


(FG C-NNS)
ACACATGGCGCAGSNNSNNACTGCTCATGCTG




GCGATCAGCTT



 


oligo 46rev
GCGGCGTTGAATCTGCACGTCCAGCTCGCCAT
455


(FG + 2
ACACATGGCGCAGSNNSNNSNNSNNACTGCTC



C-NNS)
ATGCTGGCGATCAGCTT



 


oligo 34rev
CAGGTGCACCCAGACSNNSNNCAGSNNSNNSN
456


(BC A-NNS)
NSNNSNNSNNSNNAAACTCCACGATCACAGTC




AT



 


oligo 40rev
CACTTCTTCCCACTGCACSNNSNNSNNSNNSN
457


(DE A-NNS)
NGATSNNSNNGAACATCTTCTTCATTGGGCG



 


oligo 44rev
GCGGCGTTGAATCTGCACGTCCAGCTCGCCAT
458


(FGE A-NNS)
ACACATGSNNSNNSNNSNNSNNSNNCATGCTG




GCGATCAGCTT



 


oligo 55revI
ACCATCCAGGTGCACCCAGAC A/S T/N
459


(BC A-50%
T/N G/S C/N G/N CAG G/S G/N T/N



NNS)
T/S T/N C/N T/S T/N T/N C/S A/N




G/N A/S G/N G/N A/S T/N T/N G/S




G/N T/N AAACTCCACGATCACAGTCAT



 


oligo 56revI
CACTTCTTCCCACTGCACG/S G/N T/N
460


(DE A-50%
G/S G/N A/N G/S T/N T/N T/S G/N



NNS)
G/N G/S C/N G/N GAT T/S T/N C/N




G/S C/N G/N GAACATCTTCTTCATTGGGC




G



 


oligo 57revI
GCGGCGTTGAATCTGCACGTCCAGCTCGCCAT
461


(FG A-50%
ACACATG G/S C/N G/N C/S A/N G/N



NNS)
G/S G/N A/N G/S T/N C/N A/S C/N




T/N G/S C/N T/N CATGCTGGCGATCAGC




TT



 


oligo 68revI
CAGGTGCACCCAGACATTGCGCAGSNNSNNSN
462


(BC Aext-NNS)
NSNNSNNSNNSNNAGGATTGGTAAACTCCACG




ATCACAGTCATGTC



 


oligo 69I
CTGCGCAATGTCTGGGTGCACCTGGATGGTCC
463


(Aext)
TGGAGTCACACGCCCAATGAAGAAGATGTTC



 


oligo 70revI
CACTTCTTCCCACTGCACGGTGGASNNSNNSN
464


(DE Aext-NNS)
NGATSNNSNNGAACATCTTCTTCATTGGGCG



 


oligo 72revI
AATCTGCACGTCCAGCTCGCCATACACSNNSN
465


(FG Aext-NNS)
NSNNSNNSNNSNNSNNSNNSNNSNNSNNSNNG




CTCATGCTGGCGATCAGCTT



 


oligo 66I
TAATACGACTCACTATAGGGACATCATCAACC
466


(T7Tmv Aext)
ATAACAATTACTATTTACAATTACAATGACCA




TCCCTGAGATCATC



 


oligo 73revI
TTTTTTTTTTTTTTTTTTTTAAATAGCGGATG
467


(Flag-tag)
CCTTGTCGTCGTCGTCCTTGTAGTCGCGGCGT




TGAATCTGCACGTCCAGCTCGCC



 


oligo 77I
TAATACGACTCACTATAGGGACATCATCAACC
468


(linker Aext)
ATA



 







C-terminal loop libraries









oligo 33 (AB
ATGACCATCCCTGAGATCATCATCAAGGTCCG
469


C-NNS)
CNNSNNSCAGNNSNNSGGTNNSNNSATGACTG




TGATCGTGGAGTTT



 


oligo 37 (CD
CTGCGCAATGTCTGGGTGCACCTGGATGGTNN
470


C-NNS)
SNNSGTCACACGCCCAATGAAGAAGATGTTCC




GCGAAATC



 


Oligo 43
GTGCAGTGGGAAGAAGTGTGCNNSCCANNSNN
471


(C69) (EF C-
SNNSGGGNNSCGCAAGCTGATCGCCAGCATG



NNS)




 


Oligo 38 (CD
CTGCGCAATGTCTGGGTGCACCTGGATGGTNN
472


C + 3-NNS)
SNNSNNSNNSNNSGTCACACGCCCAATGAAGA




AGATGTTCCGCGAAATC



 


oligo 32
ATGACCATCCCTGAGATCATCATCAAGGTCCG
473


(79I) (AB A-
CNNSNNSNNSNNSNNSNNSNNSNNSATGACTG



NNS)
TGATCGTGGAGTTT



 


oligo 36
CTGCGCAATGTCTGGGTGCACCTGGATNNSNN
474


(81I) (CD A-
SNNSNNSNNSNNSNNSATGAAGAAGATGTTCC



NNS)
GCGAAATC



 


oligo 83I
GTGCAGTGGGAAGAAGTGGTGNNSNNSNNSNN
475


(C69) (EF A-
SNNSNNSNNSCGCAAGCTGATCGCCAGCATGA



NNS)
GCAGT



 


oligo 84revI
GGAGCCTCCGCCTCCGCGGCGTTGAATCTGCA
476


(G4S)
CGTCCAGCTCGCCATACACATGGCGCAGGGAG




TCACTGCTCATGCTGGCGATCAG



 


oligo 85revI
TTTTTTTTTTTTTTTTTTTTAAATAGCGGATG
477


(G4S-Flag-
CCTTGTCGTCGTCGTCCTTGTAGTCGGAGCCT



tag)
CCGCCTCCGCGGCG



 







5. Primers used for construction of


factor XIIIA, F13β1, MBP domains









oligo129
AATGATCTACATATGAATGATCTAGACGTCCT
478



GGCCAAGCAAAAGTCCACCGTGCTAACCATCC




CTGAGATCATCATC



 


oligo 126 rev
TAGATCATTGGATCCCTCATTAATGATGGTGA
479



TGGTGATGCATGGAAGGGCGGCGTTGAATCTG




CACGTC



 


oligo 118
AATGATCTACATATGTCAGAAACTTCCAGGAC
480


(NdeI)
CGCCTTT



 


oligo 101 rev
CTGAAGAGATCCCTTCTGGGGTCATACGGACG
481



ACTGAAGTCAATCTGCAC



 


oligo 100
GTGCAGATTGACTTCAGTCGTCCGTATGACCC
482



CAGAAGGGATCTCTTCAG



 


oligo 103 rev
CATAGGCATAGATATTGTCCCAGGAGCCAACG
483



AGGACACCTTCGTCATC



 


oligo 102
GATGACGAAGGTGTCCTCGTTGGCTCCTGGGA
484



CAATATCTATGCCTATG



 


oligo 133 rev
ATCAGTAATATCCATCATGCCATCACCACCAA
485



TTTGTTTGGTCACAATTAAT



 


oligo 134
ATTAATTGTGACCAAACAAATTGGTGGTGATG
486



GCATGATGGATATTACTGAT



 


oligo 136 rev
CACGGTGGACTTTTGCTTGGCCAGGACGTCCC
487



TGGTCTCATTGATGCGAGCTGT



 


oligo 127
AATGATCTACATATGAAAATCGAAGAAGGTAA
488



ACTGGTA



 


oligo128 rev
TAGATCATTCATATGCCTTCCCTCGATCCCGA
489



GGTTGTT



 


oligo 132
AATGATCTACATATGTCCAACGTTGACATGGA
490


(NdeI)
CTTTGAAG



 


oligo 60
TAATACGACTCACTATAGGGACAATTACTATT
491


(NdeI)
TACAATTACATATGTCCAACGTTGACATGGAC




TTTGAA



 


Oligo 62rev
TAGATCATTGGATCCCTCATTAATGATGGTGA
492


(BamHI)
TGGTGATGTAGCACGGTGGACTTTTGCTTGGC



 


oligo EG1rev
TAGATTATTCTCGAGCTCATTATAGCACGGTG
493


(XhoI)
GACTTTTGCTTGGCCAG



 


oligo 61rev
TCCTTTGCTGAATTCGCCAGAACCAGCAGCGG
494


(BamHI)
AGCCAGCGGATCCTAGCACGGTGGACTTTTGC




TTGGC
















TABLE 11







Oligonucleotides used for ETBP phage display.


Table 11 discloses SEQ ID NOS 495-515.








Name
Sequence











FG1
ATTACGCCAAGCTTTGGAGCCTTTTTTTTGGAGATTTT



CAACGTGAAAAAATTATTATTCGCAATTCCTTTAG


 


FG2
TTATTATTCGCAATTCCTTTAGTTGTTCCTTTCTATTC



TCACTCGGCCGACGTGGCCACCATCCCTGAGATCATCA



TC


 


FG3
TGAGATGAGTTTTTGTTCTGCGGCCCCAGACGCGTGAT



GGTGATGATGATGTGCGGCCGCGCGGCGTTGAATCTGC



ACG


 


FG4
GCAGAACAAAAACTCATCTCAGAAGAGGATCTGAATGG



GGCAGCAGAAGCTAGTTCTGCTAGTGCCGAAACTGTTG



AAAGTTG


 


FG5
GTAAAACGACGGCCAGTGAATTCTCATCACGCGTTTCA



TTAAGACTCCTTATTACGCAGTATG


 


FG6
GCAGAACAAAAACTCATCTCAGAAGAGGATCTGAATGG



GGCAGCAGAAGCTAGTTCTGCTAGTGCCTCTGGTTCCG



GTGATTTTG


 


FG7
CTGAAACATGAAAGTATTAAGAGGC


 


AKx295Y
TCTATTCTCACTCGGCCGACGTGGCCGTCTTCACCATC



CCTGAGATCATCATC


 


AKx296Y
TCTATTCTCACTCGAAGACACGTGGCCACCATCCCTGA



GATCATCATC


 


AKx297Y
TGAGATGAGTTTGCGGCCGCGCGGCGTTGGTCTTCAAT



CTGCACGTCCAGCTC


 


AKx298Y
TGAGATGAGTTTGAAGACCGGCGTTGAATCTGCACGTC



CAGCTCGC


 


1867 not pI
gggatccgcggccgcaggctctaaagatatcagaactg



ttgaaagttgtttagcaaaacc


 


1868 bsu36
GGGATCCTGAGGAATTCTTATTAAGACTCCTTATTACG



CAGTATGTTAGCAA


 


1873 bgl2 b
gggagatctCCTCAGGaaccatagtacgcgccctgtag


 


1874 bsmb1
gggtgtacacgtctccggagattgtataagcaaatatt



taaattgtaaacgtt


 


1875 bsmb1
ggaattccgtctccCTCCGCCCATCCCGCCCCTAACTC



C


 


1876 H3 amp
ggaattcaagcttgggACTAGTTATGCCGAAAGGCCAT



CCTGAC


 


1877 F13 pst
ggaattccctgcaggtACCATCCCTGAGATCATCATCA



AGGT


 


1878 F13 not
GATGATGTGCGGCCGCGCGGCGTTGAATCTGCACGTCC



AGCTCGCCA


 


1882 pst bst
gagacgcacaacaccgtctcgc


 


1883 pst bst
ggccgcgagacggtgttgtgcgtctctgca
















TABLE 12







Sequence of paracompgp3 phagemid vector


LOCUS paracompgp3 4930 by








FEATURES
Location/Qualifiers





misc_binding
1 . . . 6



/dbxref = “REBASE: HindIII”


 


Other Gene
13 . . . 891



/gene = “AraC other”


 


ORF
13 . . . 942



/sequence = “ORF_3 rf(5)”


 


misc_binding
623 . . . 628



/dbxref = “REBASE: NruI”


 


Regulatory_Seq
921 . . . 936



/gene = “arao2 reg”


 


Other Gene
1120 . . . 1133



/gene = “CAP_BS other”


 


Other Gene
1130 . . . 1168



/gene = “AraI1I2 other”


 


Promoter
1165 . . . 1193



/gene = “ARA prom”


 


misc_binding
1301 . . . 1306



/dbxref = “REBASE: PstI”


 


misc_binding
1326 . . . 1333



/dbxref = “REBASE: NOtI”


 


ORF
1749 . . . 2567



/sequence = “ORF_2 rf(3)”


 


misc_binding
2241 . . . 2246



/dbxref = “REBASE: ClaI”


 


misc_binding
2571 . . . 2576



/dbxref = “REBASE: EcoRI”


 


Rep_Origin
2609 . . . 2915



/gene = “f1 origin”


 


Rep_Origin
3075 . . . 3152



/gene = “SV40 origin”


 


misc_binding
3155 . . . 3167



/dbxref = “REBASE: SfiI”


 


misc_binding
3156 . . . 3166



/dbxref = “REBASE: BglI”


 


misc_binding
3206 . . . 3211



/dbxref = “REBASE: StuI”


 


Rep_Origin
3236 . . . 3818



/gene = “pBR322 origin”


 


Marker
3893 . . . 4753



/gene = “amp marker”


 


ORF
3893 . . . 4753



/sequence = “ORF_1 rf(2)”


 


BASE COUNT 
1224 a 1206 c 1214 g 1286 t 0 others


 


ORIGIN



   1
aagcttgata tcttatgaca acttgacggc tacatcattc actttttctt cacaaccggc


 


  61
acggaactcg ctcgggctgg ccccggtgca ttttttaaat acccgcgaga aatagagttg


 


 121
atcgtcaaaa ccaacattgc gaccgacggt ggcgataggc atccgggtgg tgctcaaaag


 


 181
cagcttcgcc tggctgatac gttggtcctc gcgccagctt aagacgctaa tccctaactg


 


 241
ctggcggaaa agatgtgaca gacgcgacgg cgacaagcaa acatgctgtg cgacgctggc


 


 301
gatatcaaaa ttgctgtctg ccaggtgatc gctgatgtac tgacaagcct cgcgtacccg


 


 361
attatccatc ggtggatgga gcgactcgtt aatcgcttcc atgcgccgca gtaacaattg


 


 421
ctcaagcaga tttatcgcca gcagctccga atagcgccct tccccttgcc cggcgttaat


 


 481
gatttgccca aacaggtcgc tgaaatgcgg ctggtgcgct tcatccgggc gaaagaaccc


 


 541
cgtattggca aatattgacg gccagttaag ccattcatgc cagtaggcgc gcggacgaaa


 


 601
gtaaacccac tggtgatacc attcgcgagc ctccggatga cgaccgtagt gatgaatctc


 


 661
tcctggcggg aacagcaaaa tatcacccgg tcggcaaaca aattctcgtc cctgattttt


 


 721
caccaccccc tgaccgcgaa tggtgagatt gagaatataa cctttcattc ccagcggtcg


 


 781
gtcgataaaa aaatcgagat aaccgttggc cacaaccggc gttaaacccg ccaccagatg


 


 841
ggcattaaac gagtatcccg gcagcagggg atcattttgc gcttcagcca tacttttcat


 


 901
actcccgcca ttcagagaag aaaccaattg tccatattgc atcagacatt gccgtcactg


 


 961
cgtcttttac tggctcttct cgctaaccaa accggtaacc ccgcttatta aaagcattct


 


1021
gtaacaaagc gggaccaaag ccatgacaaa aacgcgtaac aaaagtgtct ataatcacgg


 


1081
cagaaaagtc cacattgatt atttgcacgg cgtcacactt tgctatgcca tagcattttt


 


1141
atccataaga ttagcggatc ctacctgacg ctttttatcg caactctcta ctgtttctcc


 


1201
atacccgttt ttttgggcta acaggaggaa ttaatcatga tgaagcgcaa tattctggca


 


1261
gtgatcgtcc ctgctctgtt agtagcaggt actgcaaacg ctgcagagac gcacaacacc


 


1321
gtctcgcggc cgcaggctct aaagatatca gaactgttga aagttgttta gcaaaacccc


 


1381
atacagaaaa ttcatttact aacgtctgga aagacgacaa aactttagat cgttacgcta


 


1441
actatgaggg ttgtctgtgg aatgctacag gcgttgtagt ttgtactggt gacgaaactc


 


1501
agtgttacgg tacatgggtt cctattgggc ttgctatccc tgaaaatgag ggtggtggct


 


1561
ctgagggtgg cggttctgag ggtggcggtt ctgagggtgg cggtactaaa cctcctgagt


 


1621
acggtgatac acctattccg ggctatactt atatcaaccc tctcgacggc acttatccgc


 


1681
ctggtactga gcaaaacccc gctaatccta atccttctct tgaggagtct cagcctctta


 


1741
atactttcat gtttcagaat aataggttcc gaaataggca gggggcatta actgtttata


 


1801
cgggcactgt tactcaaggc actgaccccg ttaaaactta ttaccagtac actcctgtat


 


1861
catcaaaagc catgtatgac gcttactgga acggtaaatt cagagactgc gctttccatt


 


1921
ctggctttaa tgaggatcca ttcgtttgtg aatatcaagg ccaatcgtct gacctgcctc


 


1981
aacctcctgt caatgctggc ggcggctctg gtggtggttc tggtggcggc tctgagggtg


 


2041
gtggctctga gggtggcggt tctgagggtg gcggctctga gggaggcggt tccggtggtg


 


2101
gctctggttc cggtgatttt gattatgaaa agatggcaaa cgctaataag ggggctatga


 


2161
ccgaaaatgc cgatgaaaac gcgctacagt ctgacgctaa aggcaaactt gattctgtcg


 


2221
ctactgatta cggtgctgct atcgatggtt tcattggtga cgtttccggc cttgctaatg


 


2281
gtaatggtgc tactggtgat tttgctggct ctaattccca aatggctcaa gtcggtgacg


 


2341
gtgataattc acctttaatg aataatttcc gtcaatattt accttccctc cctcaatcgg


 


2401
ttgaatgtcg cccttttgtc tttagcgctg gtaaaccata tgaattttct attgattgtg


 


2461
acaaaataaa cttattccgt ggtgtctttg cgtttctttt atatgttgcc acctttatgt


 


2521
atgtattttc tacgtttgct aacatactgc gtaataagga gtcttaataa gaattcctca


 


2581
ggaaccatag tacgcgccct gtagcggcgc attaagcgcg gcgggtgtgg tggttacgcg


 


2641
cagcgtgacc gtaacaattg ccagcgccct agcgcccgct cctttcgctt tcttcccttc


 


2701
ctttctcgcc acgttcgccg gctttccccg tcaagctcta aatcgggggc tccctttagg


 


2761
gttccgattt agtgctttac ggcacctcga ccccaaaaaa cttgatttgg gtgatggttc


 


2821
acgtagtggg ccatcgccct gatagacggt ttttcgccct ttgacgttgg agtccacgtt


 


2881
ctttaatagt ggactcttgt tccaaactgg aacaacactc aaccctatct cgggctattc


 


2941
ttttgattta taagggattt tgccgatttc ggcctattgg ttaaaaaatg agctgattta


 


3001
acaaaaattt aacgcgaatt ttaacaaaat attaacgttt acaatttaaa tatttgctta


 


3061
tacaatctcc gcccatcccg cccctaactc cgcccagttc cgcccattct ccgcctcatg


 


3121
gctgactaat tttttttatt tatgcagagg ccgaggccgc ctcggcctct gagctattcc


 


3181
agaagtagtg aggaggcttt tttggaggcc taggcttttg caaaaagcta attcggcgta


 


3241
atctgctgct tgcaaacaaa aaaaccaccg ctaccagcgg tggtttgttt gccggatcaa


 


3301
gagctaccaa ctctttttcc gaaggtaact ggcttcagca gagcgcagat accaaatact


 


3361
gtccttctag tgtagccgta gttaggccac cacttcaaga actctgtagc accgcctaca


 


3421
tacctcgctc tgctgaagcc agttaccagt ggctgctgcc agtggcgata agtcgtgtct


 


3481
taccgggttg gactcaagag atagttaccg gataaggcgc agcggtcggg ctgaacgggg


 


3541
ggttcgtgca cacagcccag cttggagcga acgacctaca ccgaactgag atacctacag


 


3601
cgtgagctat gagaaagcgc cacgcttccc gaagggagaa aggcggacag gtatccggta


 


3661
agcggcaggg tcggaacagg agagcgcacg agggagcttc cagggggaaa cgcctggtat


 


3721
ctttatagtc ctgtcgggtt tcgccacctc tgacttgagc gtcgattttt gtgatgctcg


 


3781
tcaggggggc ggagcctatg gaaaaacgcc agcaacgcaa gctagagttt aaacttgaca


 


3841
gatgagacaa taaccctgat aaatgcttca ataatattga aaaaggaaaa gtatgagtat


 


3901
tcaacatttc cgtgtcgccc ttattccctt ttttgcggca ttttgccttc ctgtttttgc


 


3961
tcacccagaa acgctggtga aagtaaaaga tgcagaagat cacttgggtg cgcgagtggg


 


4021
ttacatcgaa ctggatctca acagcggtaa gatccttgag agttttcgcc ccgaagaacg


 


4081
tttcccaatg atgagcactt ttaaagttct gctatgtggc gcggtattat cccgtattga


 


4141
tgccgggcaa gagcaactcg gtcgccgcat acactattct cagaatgact tggttgaata


 


4201
ctcaccagtc acagaaaagc atcttacgga tggcatgaca gtaagagaat tatgcagtgc


 


4261
tgccataacc atgagtgata acactgcggc caacttactt ctgacaacta tcggaggacc


 


4321
gaaggagcta accgcttttt tgcacaacat gggggatcat gtaactcgcc ttgatcgttg


 


4381
ggaaccggag ctgaatgaag ccataccaaa cgacgagcgt gacaccacga tgcctgtagc


 


4441
aatggcaaca acgttgcgaa aactattaac tggcgaacta cttactctag cttcccggca


 


4501
acaactaata gactggatgg aggcggataa agttgcagga ccacttctgc gctcggcact


 


4561
tccggctggc tggtttattg ctgataaatc aggagccggt gagcgtgggt cacgcggtat


 


4621
cattgcagca ctggggccgg atggtaagcc ctcccgtatc gtagttatct acactacggg


 


4681
gagtcaggca actatggatg aacgaaatag acagatcgct gagataggtg cctcactgat


 


4741
taagcattgg taaggataaa tttctggtaa ggaggacacg tatggaagtg ggcaagttgg


 


4801
ggaagccgta tccgttgctg aatctggcat atgtgggagt ataagacgcg cagcgtcgca


 


4861
tcaggcattt ttttctgcgc caatgcaaaa aggccatccg tcaggatggc ctttcggcat


 


4921
aactagtccc (SEQ ID NO: 516)


//
















TABLE 13







Sequence of paracompFXIIIb2pIII phagemid vector


LOCUS paracompFXIIIb2pIII 5216 bp








FEATURES
Location/Qualifiers





misc_binding
1 . . . 6



/dbxref = “REBASE: HindIII”


 


Other Gene
13 . . . 891



/gene = “AraC other”


 


ORF
13 . . . 942



/sequence = “ORF_3 rf(6)”


 


misc_binding
623 . . . 628



/dbxref = “REBASE: NruI”


 


Regulatory_Seq
921 . . . 936



/gene = “arao2 reg”


 


Other Gene
1120 . . . 1133



/gene = “CAP_BS other”


 


Other Gene
1130 . . . 1168



/gene = “AraI1I2 other”


 


Promoter
1165 . . . 1193



/gene = “ARA prom”


 


ORF
1237 . . . 2853



/sequence = “ORF_1 rf(1)”


 


misc_binding
1301 . . . 1306



/dbxref = “REBASE: PstI”


 


misc_binding
1306 . . . 1311



/dbxref = “REBASE: KpnI”


 


misc_binding
1516 . . . 1521



/dbxref = “REBASE: NcoI”


 


misc_binding
1612 . . . 1619



/dbxref = “REBASE: NotI”


 


misc_binding
2527 . . . 2532



/dbxref = “REBASE: ClaI”


 


misc_binding
2857 . . . 2862



/dbxref = “REBASE: EcoRI”


 


Rep_Origin
2895 . . . 3201



/gene = “f1 origin”


 


Rep_Origin
3361 . . . 3438



/gene = “SV40 origin”


 


misc_binding
3441 . . . 3453



/dbxref = “REBASE: SfiI”


 


misc_binding
3442 . . . 3452



/dbxref = “REBASE: BglI”


 


misc_binding
3492 . . . 3497



/dbxref = “REBASE: StuI”


 


Rep_Origin
3522 . . . 4104



/gene = “pBR322 origin”


 


Marker
4179 . . . 5039



/gene = “amp marker”


 


ORF
4179 . . . 5039



/sequence = “ORF-2 rf(3)”


 


BASE COUNT
1286 a 1285 c 1298 g 1347 t 0 others


 


ORIGIN



   1
aagcttgata tcttatgaca acttgacggc tacatcattc actttttctt cacaaccggc


 


  61
acggaactcg ctcgggctgg ccccggtgca ttttttaaat acccgcgaga aatagagttg


 


 121
atcgtcaaaa ccaacattgc gaccgacggt ggcgataggc atccgggtgg tgctcaaaag


 


 181
cagcttcgcc tggctgatac gttggtcctc gcgccagctt aagacgctaa tccctaactg


 


 241
ctggcggaaa agatgtgaca gacgcgacgg cgacaagcaa acatgctgtg cgacgctggc


 


 301
gatatcaaaa ttgctgtctg ccaggtgatc gctgatgtac tgacaagcct cgcgtacccg


 


 361
attatccatc ggtggatgga gcgactcgtt aatcgcttcc atgcgccgca gtaacaattg


 


 421
ctcaagcaga tttatcgcca gcagctccga atagcgccct tccccttgcc cggcgttaat


 


 481
gatttgccca aacaggtcgc tgaaatgcgg ctggtgcgct tcatccgggc gaaagaaccc


 


 541
cgtattggca aatattgacg gccagttaag ccattcatgc cagtaggcgc gcggacgaaa


 


 601
gtaaacccac tggtgatacc attcgcgagc ctccggatga cgaccgtagt gatgaatctc


 


 661
tcctggcggg aacagcaaaa tatcacccgg tcggcaaaca aattctcgtc cctgattttt


 


 721
caccaccccc tgaccgcgaa tggtgagatt gagaatataa cctttcattc ccagcggtcg


 


 781
gtcgataaaa aaatcgagat aaccgttggc ctcaatcggc gttaaacccg ccaccagatg


 


 841
ggcattaaac gagtatcccg gcagcagggg atcattttgc gcttcagcca tacttttcat


 


 901
actcccgcca ttcagagaag aaaccaattg tccatattgc atcagacatt gccgtcactg


 


 961
cgtcttttac tggctcttct cgctaaccaa accggtaacc ccgcttatta aaagcattct


 


1021
gtaacaaagc gggaccaaag ccatgacaaa aacgcgtaac aaaagtgtct ataatcacgg


 


1081
cagaaaagtc cacattgatt atttgcacgg cgtcacactt tgctatgcca tagcattttt


 


1141
atccataaga ttagcggatc ctacctgacg ctttttatcg caactctcta ctgtttctcc


 


1201
atacccgttt ttttgggcta acaggaggaa ttaatcatga tgaagcgcaa tattctggca


 


1261
gtgatcgtcc ctgctctgtt agtagcaggt actgcaaacg ctgcaggtac catccctgag


 


1321
atcatcatca aggtccgcgg cactcaggtc gtgggttctg acatgactgt gatcgtggag


 


1381
tttaccaatc ctctgaaaga aaccctgcgc aatgtctggg tgcacctgga tggtcctgga


 


1441
gtcacacgcc caatgaagaa gatgttccgc gaaatccgcc caaactccac cgtgcagtgg


 


1501
gaagaagtgg tccgcccatg ggtctctggg catcgcaagc tgatcgccag catgagcagt


 


1561
gactccctgc gccatgtgta tggcgagctg gacgtgcaga ttcaacgccg cgcggccgca


 


1621
ggctctaaag atatcagaac tgttgaaagt tgtttagcaa aaccccatac agaaaattca


 


1681
tttactaacg tctggaaaga cgacaaaact ttagatcgtt acgctaacta tgagggttgt


 


1741
ctgtggaatg ctacaggcgt tgtagtttgt actggtgacg aaactcagtg ttacggtaca


 


1801
tgggttccta ttgggcttgc tatccctgaa aatgagggtg gtggctctga gggtggcggt


 


1861
tctgagggtg gcggttctga gggtggcggt actaaacctc ctgagtacgg tgatacacct


 


1921
attccgggct atacttatat caaccctctc gacggcactt atccgcctgg tactgagcaa


 


1981
aaccccgcta atcctaatcc ttctcttgag gagtctcagc ctcttaatac tttcatgttt


 


2041
cagaataata ggttccgaaa taggcagggg gcattaactg tttatacggg cactgttact


 


2101
caaggcactg accccgttaa aacttattac cagtacactc ctgtatcatc aaaagccatg


 


2161
tatgacgctt actggaacgg taaattcaga gactgcgctt tccattctgg ctttaatgag


 


2221
gatccattcg tttgtgaata tcaaggccaa tcgtctgacc tgcctcaacc tcctgtcaat


 


2281
gctggcggcg gctctggtgg tggttccggt ggcggctctg agggtggtgg ctctgagggt


 


2341
ggcggttctg agggtggcgg ctctgaggga ggcggttccg gtggtggctc tggttccggt


 


2401
gattttgatt atgaaaagat ggcaaacgct aataaggggg ctatgaccga aaatgccgat


 


2461
gaaaacgcgc tacagtctga cgctaaaggc aaacttgatt ctgtcgctac tgattacggt


 


2521
gctgctatcg atggtttcat tggtgacgtt tccggccttg ctaatggtaa tggtgctact


 


2581
ggtgattttg ctggctctaa ttcccaaatg gctcaagtcg gtgacggtga taattcacct


 


2641
ttaatgaata atttccgtca atatttacct tccctccctc aatcggttga atgtcgccct


 


2701
tttgtcttta gcgctggtaa accatatgaa ttttctattg attgtgacaa aataaactta


 


2761
ttccgtggtg tctttgcgtt tcttttatat gttgccacct ttatgtatgt attttctacg


 


2821
tttgctaaca tactgcgtaa taaggagtct taataagaat tcctcaggaa ccatagtacg


 


2881
cgccctgtag cggcgcatta agcgcggcgg gtgtggtggt tacgcgcagc gtgaccgcta


 


2941
cacttgccag cgccctagcg cccgctcctt tcgctttctt cccttccttt ctcgccacgt


 


3001
tcgccggctt tccccgtcaa gctctaaatc gggggctccc tttagggttc cgatttagtg


 


3061
ctttacggca cctcgacccc aaaaaacttg atttgggtga tggttcacgt agtgggccat


 


3121
cgccctgata gacggttttt cgccctttga cgttggagtc cacgttcttt aatagtggac


 


3181
tcttgttcca aactggaaca acactcaacc ctatctcggg ctattctttt gatttataag


 


3241
ggattttgcc gatttcggcc tattggttaa aaaatgagct gatttaacaa aaatttaacg


 


3301
cgaattttaa caaaatatta acgtttacaa tttaaatatt tgcttataca atctccgccc


 


3361
atcccgcccc taactccgcc cagcttcgcc cattctccgc ctcatggctg actaattttt


 


3421
tttatttatg cagaggccga ggccgcctcg gcctctgagc tattccagaa gtagtgagga


 


3481
ggcttttttg gaggcctagg cttttgcaaa aagctaattc ggcgtaatct gctgcttgca


 


3541
aacaaaaaaa ccaccgctac cagcggtggt ttgtttgccg gatcaagagc taccaactct


 


3601
ttttccgaag gtaactggct tcagcagagc gcagatacca aatactgtcc ttctagtgta


 


3661
gccgtagtta ggccaccact tcaagaactc tgtagcaccg cctacatacc tcgctctgct


 


3721
gaagccagtt accagtggct gctgccagtg gcgataagtc gtgtcttacc gggttggact


 


3781
caagagatag ttaccggata aggcgcagcg gtcgggctga acggggggtt cgtgcacaca


 


3841
gcccagcttg gagcgaacga cctacaccga actgagatac ctacagcgtg agctatgaga


 


3901
aagcgccacg cttcccgaag ggagaaaggc ggacaggtat ccggtaagcg gcagggtcgg


 


3961
aacaggagag cgcacgaggg agcttccagg gggaaacgcc tggtatcttt atagtcctgt


 


4021
cgggtttcgc cacctctgac ttgagcgtcg atttgggtga tgctcgtcag gggggcggag


 


4081
cctatggaaa aacgccagca acgcaagcta gagtttaaac ttgacagatg agacaataac


 


4141
cctgataaat gcttcaataa tattgaaaaa ggaaaagtat gagtattcaa catttccgtg


 


4201
tcgcccttat tccctttttt gcggcatttt gccttcctgt ttttgctcac ccagaaacgc


 


4261
tggtgaaagt aaaagatgca gaagatcact tgggtgcgcg agtgggttac atcgaactgg


 


4321
atctcaacag cggtaagatc cttgagagtt ttcgccccga agaacgtttc ccaatgatga


 


4381
gcacttttaa agttctgcta tgtggcgcgg tattatcccg tattgatgcc gggcaagagc


 


4441
aactcggtcg ccgcatacac tattctcaga atgacttggt tgaatactca ccagtcacag


 


4501
aaaagcatct tacggatggc atgacagtaa gagaattatg cagtgctgcc ataaccatga


 


4561
gtgataacac tgcggccaac ttacttctga caactatcgg aggaccgaag gagctaaccg


 


4621
cttttttgca caacatgggg gatcatgtaa ctcgccttga tcgttgggaa ccggagctga


 


4681
atgaagccat accaaacgac gagcgtgaca ccacgatgcc tgtagcaatg gcaacaacgt


 


4741
tgcgaaaact attaactggc gaactactta ctctagcttc ccggcaacaa ctaatagact


 


4801
ggatggaggc ggataaagtt gcaggaccac ttctgcgctc ggcacttccg gctggctggt


 


4861
ttattgctga taaatcagga gccggtgagc gtgggtcacg cggtatcatt gcagcactgg


 


4921
ggccggatgg taagccctcc cgtatcgtag ttatctacac tacggggagt caggcaacta


 


4981
tggatgaacg aaatagacag atcgctgaga taggtgcctc actgattaag cattggtaag


 


5041
gataaatttc tggtaaggag gacacgtatg gaagtgggca agttggggaa gccgtatccg


 


5101
ttgctgaatc tggcatatgt gggagtataa gacgcgcagc gtcgcatcag gcattttttt


 


5161
ctgcgccaat gcaaaaaggc catccgtcag gatggccttt cggcataact agtccc (SEQ ID



NO: 517)


//
















TABLE 14







Sequence of pET28_wtF13-his


LOCUS pET28-wtF13-his 5565 bp








FEATURES
Location/Qualifiers





Terminator
1 . . . 129



/gene = “T7 term”


 


misc_binding
158 . . . 163



/dbxref = “REBASE: XhoI”


 


misc_binding
161 . . . 166



/dbxref = “REBASE: SacI”


 


misc_binding
403 . . . 408



/dbxref = “REBASE: DraI”


 


misc_binding
492 . . . 497



/dbxref = “REBASE: NdeI”


 


misc_binding
531 . . . 536



/dbxref = “REBASE: XbaI”


 


Regulatory_Seq
537 . . . 564



/gene = “lac0 reg”


 


Promoter
564 . . . 582



/gene = “T7 prom”


 


misc_binding
597 . . . 602



/dbxref = “REBASE: BglII”


 


misc_binding
790 . . . 795



/dbxref = “REBASE: SphI”


 


Regulatory_Seq
960 . . . 2051



/gene = “lacI reg”


 


ORF
1092 . . . 2051



/sequence = “ORF_1 rf(3)”


 


misc_binding
1333 . . . 1338



/dbxref = “REBASE: BclI”


 


misc_binding
1526 . . . 1531



/dbxref = “REBASE: ApaI”


 


misc_binding
1767 . . . 1772



/dbxref = “REBASE: EcoRV”


 


misc_binding
1823 . . . 1828



/dbxref = “REBASE: HpaI”


 


misc_binding
1823 . . . 1828



/dbxref = “REBASE: HincII”


 


misc_binding
2377 . . . 2387



/dbxref = “REBASE: BglI”


 


Other Gene
2860 . . . 3051



/gene = “ROP other”


 


misc_binding
3189 . . . 3194



/dbxref = “REBASE: AccI”


 


Rep_Origin
3466 . . . 4085



/gene = “pBR322 origin”


 


misc_binding
3831 . . . 3839



/dbxref = “REBASE: AlwNI”


 


Marker
4191 . . . 5006



/gene = “kan2 marker”


 


ORF
4191 . . . 5006



/sequence = “ORF_2 rf(3)”


 


misc_binding
4277 . . . 4282



/dbxref = “REBASE: NruI” 


 


misc_binding
4312 . . . 4317



/dbxref = “REBASE: ClaI”


 


misc_binding
4494 . . . 4499



/dbxref = “REBASE: SmaI”


 


misc_binding
4494 . . . 4499



/dbxref = “REBASE: XmaI”


 


Rep_Origin
5231 . . . 5537



/gene = “f1 origin”


 


BASE COUNT
1320 a 1437 c 1481 g 1327 t 0 others


 


ORIGIN



   1
atccggatat agttcctcct ttcagcaaaa aacccctcaa gacccgttta gaggccccaa


  61
ggggttatgc tagttattgc tcagcggtgg cagcagccaa ctcagcttcc tttcgggctt


 


 121
tgttagcagc cggatctcag tggtggtggt ggtggtgctc gagctcatta atgatggtga


 


 181
tggtgatgtc gtctttgaat ctgcacgtcc agctcgccat acacatgtct cagggagtca


 


 241
ctgctcatgc tggctatcag cttccgatgc ccagagaccc agggccggca cacttcttcc


 


 301
cactgcacgg tggagttggg ccggatttca cggaacatct tcttcattgg tcttgttact


 


 361
ccaggaccat ccaggtgtac ccagacattt cgcagggttt cttttaaagg attggtaaac


 


 421
tcaactatca cagtcatgtc agaaccaact acctgagtgc cacggacctt gatgatgatc


 


 481
tcagggatgg tcatatgtat atctccttct taaagttaaa caaaattatt tctagagggg


 


 541
aattgttatc cgctcacaat tcccctatag tgagtcgtat taatttcgcg ggatcgagat


 


 601
ctcgatcctc tacgccggac gcatcgtggc cggcatcacc ggcgccacag gtgcggttgc


 


 661
tggcgcctat atcgccgaca tcaccgatgg ggaagatcgg gctcgccact tcgggctcat


 


 721
gagcgcttgt ttcggcgtgg gtatggtggc aggccccgtg gccgggggac tgttgggcgc


 


 781
catctccttg catgcaccat tccttgcggc ggcggtgctc aacggcctca acctactact


 


 841
gggctgcttc ctaatgcagg agtcgcataa gggagagcgt cgagatcccg gacaccatcg


 


 901
aatggcgcaa aacctttcgc ggtatggcat gatagcgccc ggaagagagt caattcaggg


 


 961
tggtgaatgt gaaaccagta acgttatacg atgtcgcaga gtatgccggt gtctcttatc


 


1021
agaccgtttc ccgcgtggtg aaccaggcca gccacgtttc tgcgaaaacg cgggaaaaag


 


1081
tggaagcggc gatggcggag ctgaattaca ttcccaaccg cgtggcacaa caactggcgg


 


1141
gcaaacagtc gttgctgatt ggcgttgcca cctccagtct ggccctgcac gcgccgtcgc


 


1201
aaattgtcgc ggcgattaaa tctcgcgccg atcaactggg tgccagcgtg gtggtgtcga


 


1261
tggtagaacg aagcggcgtc gaagcctgta aagcggcggt gcacaatctt ctcgcgcaac


 


1321
gcgtcagtgg gctgatcatt aactatccgc tggatgacca ggatgccatt gctgtggaag


 


1381
ctgcctgcac taatgttccg gcgttatttc ttgatgtctc tgaccagaca cccatcaaca


 


1441
gtattatttt ctcccatgaa gacggtacgc gactgggcgt ggagcatctg gtcgcattgg


 


1501
gtcaccagca aatcgcgctg ttagcgggcc cattaagttc tgtctcggcg cgtctgcgtc


 


1561
tggctggctg gcataaatat ctcactcgca atcaaattca gccgatagcg gaacgggaag


 


1621
gcgactggag tgccatgtcc ggttttcaac aaaccatgca aatgctgaat gagggcatcg


 


1681
ttcccactgc gatgctggtt gccaacgatc agatggcgct gggcgcaatg cgcgccatta


 


1741
ccgagtccgg gctgcgcgtt ggtgcggata tctcggtagt gggatacgac gataccgaag


 


1801
acagctcatg ttatatcccg ccgttaacca ccatcaaaca ggattttcgc ctgctggggc


 


1861
aaaccagcgt ggaccgcttg ctgcaactct ctcagggcca ggcggtgaag ggcaatcagc


 


1921
tgttgcccgt ctcactggtg aaaagaaaaa ccaccctggc gcccaatacg caaaccgcct


 


1981
ctccccgcgc gttggccgat tcattaatgc agctggcacg acaggtttcc cgactggaaa


 


2041
gcgggcagtg agcgcaacgc aattaatgta agttagctca ctcattaggc accgggatct


 


2101
cgaccgatgc ccttgagagc cttcaaccca gtcagctcct tccggtgggc gcggggcatg


 


2161
actatcgtcg ccgcacttat gactgtcttc tttatcatgc aactcgtagg acaggtgccg


 


2221
gcagcgctct gggtcatttt cggcgaggac cgctttcgct ggagcgcgac gatgatcggc


 


2281
ctgtcgcttg cggtattcgg aatcttgcac gccctcgctc aagccttcgt cactggtccc


 


2341
gccaccaaac gtttcggcga gaagcaggcc attatcgccg gcatggcggc cccacgggtg


 


2401
cgcatgatcg tgctcctgtc gttgaggacc cggctaggct ggcggggttg ccttactggt


 


2461
tagcagaatg aatcaccgat acgcgagcga acgtgaagcg actgctgctg caaaacgtct


 


2521
gcgacctgag caacaacatg aatggtcttc ggtttccgtg tttcgtaaag tctggaaacg


 


2581
cggaagtcag cgccctgcac cattatgttc cggatctgca tcgcaggatg ctgctggcta


 


2641
ccctgtggaa cacctacatc tgtattaacg aagcgctggc attgaccctg agtgattttt


 


2701
ctctggtccc gccgcatcca taccgccagt tgtttaccct cacaacgttc cagtaaccgg


 


2761
gcatgttcat catcagtaac ccgtatcgtg agcatcctct ctcgtttcat cggtatcatt


 


2821
acccccatga acagaaatcc cccttacacg gaggcatcag tgaccaaaca ggaaaaaacc


 


2881
gcccttaaca tggcccgctt tatcagaagc cagacattaa cgcttctgga gaaactcaac


 


2941
gagctggacg cggatgaaca ggcagacatc tgtgaatcgc ttcacgacca cgctgatgag


 


3001
ctttaccgca gctgcctcgc gcgtttcggt gatgacggtg aaaacctctg acacatgcag


 


3061
ctcccggaga cggtcacagc ttgtctgtaa gcggatgccg ggagcagaca agcccgtcag


 


3121
ggcgcgtcag cgggtgttgg cgggtgtcgg ggcgcagcca tgacccagtc acgtagcgat


 


3181
agcggagtgt atactggctt aactatgcgg catcagagca gattgtactg agagtgcacc


 


3241
atatatgcgg tgtgaaatac cgcacagatg cgtaaggaga aaataccgca tcaggcgctc


 


3301
ttccgcttcc tcgctcactg actcgctgcg ctcggtcgtt cggctgcggc gagcggtatc


 


3361
agctcactca aaggcggtaa tacggttatc cacagaatca ggggataacg caggaaagaa


 


3421
catgtgagca aaaggccagc aaaaggccag gaaccgtaaa aaggccgcgt tgctggcgtt


 


3481
tttccatagg ctccgccccc ctgacgagca tcacaaaaat cgacgctcaa gtcagaggtg


 


3541
gcgaaacccg acaggactat aaagatacca ggcgtttccc cctggaagct ccctcgtgcg


 


3601
ctctcctgtt ccgaccctgc cgcttaccgg atacctgtcc gcctttctcc cttcgggaag


 


3661
cgtggcgctt tctcatagct cacgctgtag gtatctcagt tcggtgtagg tcgttcgctc


 


3721
caagctgggc tgtgtgcacg aaccccccgt tcagcccgac cgctgcgcct tatccggtaa


 


3781
ctatcgtctt gagtccaacc cggtaagaca cgacttatcg ccactggcag cagccactgg


 


3841
taacaggatt agcagagcga ggtatgtagg cggtgctaca gagttcttga agtggtggcc


 


3901
taactacggc tacactagaa ggacagtatt tggtatctgc gctctgctga agccagttac


 


3961
cttcggaaaa agagttggta gctcttgatc cggcaaacaa accaccgctg gtagcggtgg


 


4021
tttttttgtt tgcaagcagc agattacgcg cagaaaaaaa ggatctcaag aagatccttt


 


4081
gatcttttct acggggtctg acgctcagtg gaacgaaaac tcacgttaag ggattttggt


 


4141
catgaacaat aaaactgtct gcttacataa acagtaatac aaggggtgtt atgagccata


 


4201
ttcaacggga aacgtcttgc tctaggccgc gattaaattc caacatggat gctgatttat


 


4261
atgggtataa atgggctcgc gataatgtcg ggcaatcagg tgcgacaatc tatcgattgt


 


4321
atgggaagcc cgatgcgcca gagttgtttc tgaaacatgg caaaggtagc gttgccaatg


 


4381
atgttacaga tgagatggtc agactaaact ggctgacgga atttatgcct cttccgacca


 


4441
tcaagcattt tatccgtact cctgatgatg catggttact caccactgcg atccccggga


 


4501
aaacagcatt ccaggtatta gaagaatatc ctgattcagg tgaaaatatt gttgatgcgc


 


4561
tggcagtgtt cctgcgccgg ttgcattcga ttcctgtttg taattgtcct tttaacagcg


 


4621
atcgcgtatt tcgtctcgct caggcgcaat cacgaatgaa taacggtttg gttgatgcga


 


4681
gtgattttga tgacgagcgt aatggctggc ctgttgaaca agtctggaaa gaaatgcata


 


4741
aacttttgcc attctcaccg gattcagtcg tcactcatgg tgatttctca cttgataacc


 


4801
ttatttttga cgaggggaaa ttaataggtt gtattgatgt tggacgagtc ggaatcgcag


 


4861
accgatacca ggatcttgcc atcctatgga actgcctcgg tgagttttct ccttcattac


 


4921
agaaacggct ttttcaaaaa tatggtattg ataatcctga tatgaataaa ttgcagtttc


 


4981
atttgatgct cgatgagttt ttctaagaat taattcatga gcggatacat atttgaatgt


 


5041
atttagaaaa ataaacaaat aggggttccg cgcacatttc cccgaaaagt gccacctgaa


 


5101
attgtaaacg ttaatatttt gttaaaattc gcgttaaatt tttgttaaat cagctcattt


 


5161
tttaaccaat aggccgaaat cggcaaaatc ccttataaat caaaagaata gaccgagata


 


5221
gggttgagtg ttgttccagt ttggaacaag agtccactat taaagaacgt ggactccaac


 


5281
gtcaaagggc gaaaaaccgt ctatcagggc gatggcccac tacgtgaacc atcaccctaa


 


5341
tcaagttttt tggggtcgag gtgccgtaaa gcactaaatc ggaaccctaa agggagcccc


 


5401
cgatttagag cttgacgggg aaagccggcg aacgtggcga gaaaggaagg gaagaaagcg


 


5461
aaaggagcgg gcgctagggc gctggcaagt gtagcggtca cgctgcgcgt aaccaccaca


 


5521
cccgccgcgc ttaatgcgcc gctacagggc gcgtcccatt cgcca (SEQ ID NO: 518)


//
















TABLE 15







Sequence of FXIIIb2BbsIpIIICT


LOCUS paracompFXIIIb2BbsIpIIICT 4111 bp








FEATURES
Location/Qualifiers





Promoter
143 . . . 172



/gene = “lac prom”


 


ORF
217 . . . 1218



/sequence = “ORF_1 rf(1)”


 


misc_binding
234 . . . 239



/dbxref = “REBASE: HindIII”


 


misc_binding
321 . . . 333



/dbxref = “REBASE: SfiI”


 


misc_binding
649 . . . 656



/dbxref = “REBASE: NotI”


 


Tag
691 . . . 720



/gene = “c_myc tag”


 


misc_binding
892 . . . 897



/dbxref = “REBASE: ClaI”


 


misc_binding
1088 . . . 1093



/dbxref = “REBASE: NdeI”


 


misc_binding
1234 . . . 1239



/dbxref = “REBASE: EcoRI”


 


Reporter
1237 . . . 1396



/gene = “lacZ_a reporter”


 


misc_binding
1395 . . . 1400



/dbxref = “REBASE: NarI”


 


Rep_Origin
1486 . . . 1792



/gene = “f1 origin”


 


misc_binding
1806 . . . 1811



/dbxref = “REBASE: AvrI”


 


misc_binding
1806 . . . 1811



/dbxref = “REBASE: AvaI”


 


Promoter
2241 . . . 2269



/gene = “amp prom”


 


Marker
2311 . . . 3171



/gene = “amp marker”


 


ORF
2311 . . . 3171



/sequence = “ORF_2 rf(1)”


 


Rep_Origin
3326 . . . 3945



/gene = “pBR322 origin”


 


misc_binding
3572 . . . 3580



/dbxref = “REBASE: AlwNI”


 


BASE COUNT
999 a 1017 c 1003 g 1092 t 0 others


 


ORIGIN



   1
agcgcccaat acgcaaaccg cctctccccg cgcgttggcc gattcattaa tgcagctggc


 


  61
acgacaggtt tcccgactgg aaagcgggca gtgagcgcaa cgcaattaat gtgagttagc


 


 121
tcactcatta ggcaccccag gctttacact ttatgcttcc ggctcgtatg ttgtgtggaa


 


 181
ttgtgagcgg ataacaattt cacacaggaa acagctatga ccatgattac gccaagcttt


 


 241
ggagcctttt ttttggagat tttcaacgtg aaaaaattat tattcgcaat tcctttagtt


 


 301
gttcctttct attctcactc ggccgacgtg gccgtcttca ccatccctga gatcatcatc


 


 361
aaggtccgtg gcactcaggt agttggttct gacatgactg tgatagttga gtttaccaat


 


 421
cctttaaaag aaaccctgcg aaatgtctgg gtacacctgg atggtcctgg agtaacaaga


 


 481
ccaatgaaga agatgttccg tgaaatccgg cccaactcca ccgtgcagtg ggaagaagtg


 


 541
tgccggccct gggtctctgg gcatcggaag ctgatagcca gcatgagcag tgactccctg


 


 601
agacatgtgt atggcgagct ggacgtgcag attgaagacc aacgccgcgc ggccgcacat


 


 661
catcatcacc atcacgcgtc tggggccgca gaacaaaaac tcatctcaga agaggatctg


 


 721
aatggggcag cagaagctag ttctgctagt gcctctggtt ccggtgattt tgattatgaa


 


 781
aagatggcaa acgctaataa gggggctatg accgaaaatg ccgatgaaaa cgcgctacag


 


 841
tctgacgcta aaggcaaact tgattctgtc gctactgatt acggtgctgc tatcgatggt


 


 901
ttcattggtg acgtttccgg ccttgctaat ggtaatggtg ctactggtga ttttgctggc


 


 961
tctaattccc aaatggctca agtcggtgac ggtgataatt cacctttaat gaataatttc


 


1021
cgtcaatatt taccttccct ccctcaatcg gttgaatgtc gcccttttgt ctttagcgct


 


1081
ggtaaaccat atgaattttc tattgattgt gacaaaataa acttattccg tggtgtcttt


 


1141
gctattcttt tatatgttgc cacctttatg tatgtatttt ctacgtttgc taacatactg


 


1201
cgtaataagg agtcttaatg aaacgcgtga tgagaattca ctggccgtcg ttttacaacg


 


1261
tcgtgactgg gaaaaccctg gcgttaccca acttaatcgc cttgcagcac atcccccttt


 


1321
cgccagctgg cgtaatagcg aagaggcccg caccgatcgc ccttcccaac agttgcgcag


 


1381
cctgaatggc gaatggcgcc tgatgcggta ttttctcctt acgcatctgt gcggtatttc


 


1441
acaccgcata cgtcaaagca accatagtac gcgccctgta gcggcgcatt aagcgcggcg


 


1501
ggtgtggtgg ttacgcgcag cgtgaccgct acacttgcca gcgccctagc gcccgctcct


 


1561
ttcgctttct tcccttcctt tctcgccacg ttcgccggct ttccccgtca agctctaaat


 


1621
cgggggctcc ctttagggtt ccgatttagt gctttacggc acctcgaccc caaaaaactt


 


1681
gatttgggtg atggttcacg tagtgggcca tcgccctgat agacggtttt tcgccctttg


 


1741
acgttggagt ccacgttctt taatagtgga ctcttgttcc aaactggaac aacactcaac


 


1801
cctatctcgg gctattcttt tgatttataa gggattttgc cgatttcggc ctattggtta


 


1861
aaaaatgagc tgatttaaca aaaatttaac gcgaatttta acaaaatatt aacgtttaca


 


1921
attttatggt gcactctcag tacaatctgc tctgatgccg catagttaag ccagccccga


 


1981
cacccgccaa cacccgctga cgcgccctga cgggcttgtc tgctcccggc atccgcttac


 


2041
agacaagctg tgaccgtctc cgggagctgc atgtgtcaga ggttttcacc gtcatcaccg


 


2101
aaacgcgcga gacgaaaggg cctcgtgata cgcctatttt tataggttaa tgtcatgata


 


2161
ataatggttt cttagacgtc aggtggcact tttcggggaa atgtgcgcgg aacccctatt


 


2221
tgtttatttt tctaaataca ttcaaatatg tatccgctca tgagacaata accctgataa


 


2281
atgcttcaat aatattgaaa aaggaagagt atgagtattc aacatttccg tgtcgccctt


 


2341
attccctttt ttgcggcatt ttgccttcct gtttttgctc acccagaaac gctggtgaaa


 


2401
gtaaaagatg ctgaagatca gttgggtgca cgagtgggtt acatcgaact ggatctcaac


 


2461
agcggtaaga tccttgagag ttttcgcccc gaagaacgtt ttccaatgat gagcactttt


 


2521
aaagttctgc tatgtggcgc ggtattatcc cgtattgacg ccgggcaaga gcaactcggt


 


2581
cgccgcatac actattctca gaatgacttg gttgagtact caccagtcac agaaaagcat


 


2641
cttacggatg gcatgacagt aagagaatta tgcagtgctg ccataaccat gagtgataac


 


2701
actgcggcca acttacttct gacaacgatc ggaggaccga aggagctaac cgcttttttg


 


2761
cacaacatgg gggatcatgt aactcgcctt gatcgttggg aaccggagct gaatgaagcc


 


2821
ataccaaacg acgagcgtga caccacgatg cctgtagcaa tggcaacaac gttgcgcaaa


 


2881
ctattaactg gcgaactact tactctagct tcccggcaac aattaataga ctggatggag


 


2941
gcggataaag ttgcaggacc acttctgcgc tcggcccttc cggctggctg gtttattgct


 


3001
gataaatctg gagccggtga gcgtgggtct cgcggtatca ttgcagcact ggggccagat


 


3061
ggtaagccct cccgtatcgt agttatctac acgacgggga gtcaggcaac tatggatgaa


 


3121
cgaaatagac agatcgctga gataggtgcc tcactgatta agcattggta actgtcagac


 


3181
caagtttact catatatact ttagattgat ttaaaacttc atttttaatt taaaaggatc


 


3241
taggtgaaga tcctttttga taatctcatg accaaaatcc cttaacgtga gttttcgttc


 


3301
cactgagcgt cagaccccgt agaaaagatc aaaggatctt cttgagatcc tttttttctg


 


3361
cgcgtaatct gctgcttgca aacaaaaaaa ccaccgctac cagcggtggt ttgtttgccg


 


3421
gatcaagagc taccaactct ttttccgaag gtaactggct tcagcagagc gcagatacca


 


3481
aatactgtcc ttctagtgta gccgtagtta ggccaccact tcaagaactc tgtagcaccg


 


3541
cctacatacc tcgctctgct aatcctgtta ccagtggctg ctgccagtgg cgataagtcg


 


3601
tgtcttaccg ggttggactc aagacgatag ttaccggata aggcgcagcg gtcgggctga


 


3661
acggggggtt cgtgcacaca gcccagcttg gagcgaacga cctacaccga actgagatac


 


3721
ctacagcgtg agctatgaga aagcgccacg cttcccgaag ggagaaaggc ggacaggtat


 


3781
ccggtaagcg gcagggtcgg aacaggagag cgcacgaggg agcttccagg gggaaacgcc


 


3841
tggtatcttt atagtcctgt cgggtttcgc cacctctgac ttgagcgtcg atttttgtga


 


3901
tgctcgtcag gggggcggag cctatggaaa aacgccagca acgcggcctt tttacggttc


 


3961
ctggcctttt gctggccttt tgctcacatg ttctttcctg cgttatcccc tgattctgtg


 


4021
gataaccgta ttaccgcctt tgagtgagct gataccgctc gccgcagccg aacgaccgag


 


4081
cgcagcgagt cagtgagcga ggaagcggaa g (SEQ ID NO: 519)


//
















TABLE 16







Sequence of pF13wt-modstoppIIICT


LOCUS pFl3wt-modstoppIIICT 4099 bp








FEATURES
Location/Qualifiers





Promoter
143 . . . 172



/gene = “lac prom”


 


misc_binding
234 . . . 239



/dbxref = “REBASE: HindIII”


 


misc_binding
321 . . . 333



/dbxref = “REBASE: SfiI”


 


misc_binding
541 . . . 546



/dbxref = “REBASE: NcoI”


 


misc_binding
637 . . . 644



/dbxref = “REBASE: NotI”


 


Tag
679 . . . 708



/gene = “c_myc tag”


 


misc_binding
880 . . . 885



/dbxref = “REBASE: ClaI”


 


misc_binding
1076 . . . 1081



/dbxref = “REBASE: NdeI”


 


misc_binding
1222 . . . 1227



/dbxref = “REBASE: EcoRI”


 


Reporter
1225 . . . 1384



/gene = “lacZ_a reporter”


 


misc_binding
1383 . . . 1388



/dbxref = “REBASE: NarI”


 


misc_binding
1552 . . . 1557



/dbxref = “REBASE: AvrI”


 


misc_binding
1552 . . . 1557



/dbxref = “REBASE: AvaI”


 


Rep_Origin
1571 . . . 1877



/gene = “f1 origin”


 


Promoter
2229 . . . 2257



/gene = “amp prom”


 


Marker
2299 . . . 3159



/gene = “amp marker”


 


ORF
2299 . . . 3159



/sequence = “ORF_1 rf(1)”


 


Rep_Origin
3314 . . . 3933



/gene = “pBR322 origin”


 


misc_binding
3560 . . . 3568



/dbxref = “REBASE: AlwNI”


 


BASE COUNT
1027 a 1004 c 1025 g 1043 t 0 others


 


ORIGIN



   1
agcgcccaat acgcaaaccg cctctccccg cgcgttggcc gattcattaa tgcagctggc


 


  61
acgacaggtt tcccgactgg aaagcgggca gtgagcgcaa cgcaattaat gtgagttagc


 


 121
tcactcatta ggcaccccag gctttacact ttatgcttcc ggctcgtatg ttgtgtggaa


 


 181
ttgtgagcgg ataacaattt cacacaggaa acagctatga ccatgattac gccaagcttt


 


 241
ggagcctttt ttttggagat tttcaacgtg aaaaaattat tattcgcaat tcctttagtt


 


 301
gttcctttct attctcactc ggccgacgtg gccaccatcc ctgagatcat catcaaggtc


 


 361
cgcgggaccc aggtcgtggg ttctgacatg actgtgatcg tggagtttac caatcctctg


 


 421
taagaaaccc tgcgcaatgt ctgggtgcac ctggacggtc cgggggtgac ccgcccaatg


 


 481
aagaagatgt tccgcgaaat ccgcccaaac taaaccgtgc agtgggaaga agtggtccgc


 


 541
ccatgggtct ctggtcaccg caagctgatc gccagcatga gcagtgacta actgcgccat


 


 601
gtgtatggcg agctggacgt gcagattcaa cgccgcgcgg ccgcacatca tcatcaccat


 


 661
cacgcgtctg gggccgcaga acaaaaactc atctcagaag aggatctgaa tggggcagca


 


 721
gaagctagtt ctgctagtgc ctctggttcc ggtgattttg attatgaaaa gatggcaaac


 


 781
gctaataagg gggctatgac cgaaaatgcc gatgaaaacg cgctacagtc tgacgctaaa


 


 841
ggcaaacttg attctgtcgc tactgattac ggtgctgcta tcgatggttt cattggtgac


 


 901
gtttccggcc ttgctaatgg taatggtgct actggtgatt ttgctggctc taattcccaa


 


 961
atggctcaag tcggtgacgg tgataattca cctttaatga ataatttccg tcaatattta


 


1021
ccttccctcc ctcaatcggt tgaatgtcgc ccttttgtct ttagcgctgg taaaccatat


 


1081
gaattttcta ttgattgtga caaaataaac ttattccgtg gtgtctttgc gtttctttta


 


1141
tatgttgcca cctttatgta tgtattttct acgtttgcta acatactgcg taataaggag


 


1201
tcttaatgaa acgcgtgatg agaattcact ggccgtcgtt ttacaacgtc gtgactggga


 


1261
aaaccctggc gttacccaac ttaatcgcct tgcagcacat ccccctttcg ccagctggcg


 


1321
taatagcgaa gaggcccgca ccgatcgccc ttcccaacag ttgcgcagcc tgaatggcga


 


1381
atggcgcctg atgcggtatt ttctccttac gcatctgtgc ggtatttcac accgcataaa


 


1441
attgtaaacg ttaatatttt gttaaaattc gcgttaaatt tttgttaaat cagctcattt


 


1501
tttaaccaat aggccgaaat cggcaaaatc ccttataaat caaaagaata gcccgagata


 


1561
gggttgagtg ttgttccagt ttggaacaag agtccactat taaagaacgt ggactccaac


 


1621
gtcaaagggc gaaaaaccgt ctatcagggc gatggcccac tacgtgaacc atcacccaaa


 


1681
tcaagttttt tggggtcgag gtgccgtaaa gcactaaatc ggaaccctaa agggagcccc


 


1741
cgatttagag cttgacgggg aaagccggcg aacgtggcga gaaaggaagg gaagaaagcg


 


1801
aaaggagcgg gcgctagggc gctggcaagt gtagcggtca cgctgcgcgt aaccaccaca


 


1861
cccgccgcgc ttaatgcgcc gctacagggc gcgtactatg gttgctttga cgtatggtgc


 


1921
actctcagta caatctgctc tgatgccgca tagttaagcc agccccgaca cccgccaaca


 


1981
cccgctgacg cgccctgacg ggcttgtctg ctcccggcat ccgcttacag acaagctgtg


 


2041
accgtctccg ggagctgcat gtgtcagagg ttttcaccgt catcaccgaa acgcgcgaga


 


2101
cgaaagggcc tcgtgatacg cctattttta taggttaatg tcatgataat aatggtttct


 


2161
tagacgtcag gtggcacttt tcggggaaat gtgcgcggaa cccctatttg tttatttttc


 


2221
taaatacatt caaatatgta tccgctcatg agacaataac cctgataaat gcttcaataa


 


2281
tattgaaaaa ggaagagtat gagtattcaa catttccgtg tcgcccttat tccctttttt


 


2341
gcggcatttt gccttcctgt ttttgctcac ccagaaacgc tggtgaaagt aaaagatgct


 


2401
gaagatcagt tgggtgcacg agtgggttac atcgaactgg atctcaacag cggtaagatc


 


2461
cttgagagtt ttcgccccga agaacgtttt ccaatgatga gcacttttaa agttctgcta


 


2521
tgtggcgcgg tattatcccg tattgacgcc gggcaagagc aactcggtcg ccgcatacac


 


2581
tattctcaga atgacttggt tgagtactca ccagtcacag aaaagcatct tacggatggc


 


2641
atgacagtaa gagaattatg cagtgctgcc ataaccatga gtgataacac tgcggccaac


 


2701
ttacttctga caacgatcgg aggaccgaag gagctaaccg cttttttgca caacatgggg


 


2761
gatcatgtaa ctcgccttga tcgttgggaa ccggagctga atgaagccat accaaacgac


 


2821
gagcgtgaca ccacgatgcc tgtagcaatg gcaacaacgt tgcgcaaact attaactggc


 


2881
gaactactta ctctagcttc ccggcaacaa ttaatagact ggatggaggc ggataaagtt


 


2941
gcaggaccac ttctgcgctc ggcccttccg gctggctggt ttattgctga taaatctgga


 


3001
gccggtgagc gtgggtctcg cggtatcatt gcagcactgg ggccagatgg taagccctcc


 


3061
cgtatcgtag ttatctacac gacggggagt caggcaacta tggatgaacg aaatagacag


 


3121
atcgctgaga taggtgcctc actgattaag cattggtaac tgtcagacca agtttactca


 


3181
tatatacttt agattgattt aaaacttcat ttttaattta aaaggatcta ggtgaagatc


 


3241
ctttttgata atctcatgac caaaatccct taacgtgagt tttcgttcca ctgagcgtca


 


3301
gaccccgtag aaaagatcaa aggatcttct tgagatcctt tttttctgcg cgtaatctgc


 


3361
tgcttgcaaa caaaaaaacc accgctacca gcggtggttt gtttgccgga tcaagagcta


 


3421
ccaactcttt ttccgaaggt aactggcttc agcagagcgc agataccaaa tactgtcctt


 


3481
ctagtgtagc cgtagttagg ccaccacttc aagaactctg tagcaccgcc tacatacctc


 


3541
gctctgctaa tcctgttacc agtggctgct gccagtggcg ataagtcgtg tcttaccggg


 


3601
ttggactcaa gacgatagtt accggataag gcgcagcggt cgggctgaac ggggggttcg


 


3661
tgcacacagc ccagcttgga gcgaacgacc tacaccgaac tgagatacct acagcgtgag


 


3721
ctatgagaaa gcgccacgct tcccgaaggg agaaaggcgg acaggtatcc ggtaagcggc


 


3781
agggtcggaa caggagagcg cacgagggag cttccagggg gaaacgcctg gtatctttat


 


3841
agtcctgtcg ggtttcgcca cctctgactt gagcgtcgat ttttgtgatg ctcgtcaggg


 


3901
gggcggagcc tatggaaaaa cgccagcaac gcggcctttt tacggttcct ggccttttgc


 


3961
tggccttttg ctcacatgtt ctttcctgcg ttatcccctg attctgtgga taaccgtatt


 


4021
accgcctttg agtgagctga taccgctcgc cgcagccgaa cgaccgagcg cagcgagtca


 


4081
gtgagcgagg aagcggaag (SEQ ID NO: 520)


//









Other Embodiments
Various modifications and variations of the described methods and compositions 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 desired 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 fields of medicine, immunology, pharmacology, endocrinology, or related fields are intended to be within the scope of the invention.
All publications mentioned in this specification are herein incorporated by reference to the same extent as if each independent publication was specifically and individually incorporated by reference.

## Claims

1. An engineered Factor XIII barrel protein comprising a Factor XIII β2 barrel protein domain comprising:
seven scaffold regions, and
six intervening regions,
wherein said seven scaffold regions consist of peptides having the sequences of SEQ ID NOS: 658-664, respectively, and at least one of said intervening regions is mutated to have a loop-diversified region having less than 30% sequence identity to SEQ ID NO: 595, and wherein the protein has a beta barrel structure to increase thermostability.

2. The engineered Factor XIII barrel protein of claim 1, wherein said barrel protein is covalently linked to a polyethylene glycol (PEG).

3. The engineered Factor XIII barrel protein of claim 2, wherein said PEG is a branched PEG.

4. The engineered Factor XIII barrel protein of claim 2, wherein said barrel protein comprises a single cysteine and said cysteine is covalently bound to said PEG.

5. The engineered Factor XIII barrel protein of claim 1, wherein said engineered barrel protein is a human Factor XIII barrel protein.

6. The engineered Factor XIII barrel protein of claim 1, wherein said barrel protein is conjugated to albumin.

7. The engineered Factor XIII barrel protein of claim 1, wherein at least two of said intervening regions has an amino acid sequence of no greater than 30% identity to SEQ ID NO: 595.

8. The engineered Factor XIII barrel protein of claim 7, wherein at least three of said intervening regions has an amino acid sequence of no greater than 30% identity to SEQ ID NO: 595.

9. The engineered Factor XIII barrel protein of claim 1, which has a melting temperature (Tm) of at least 55° C.

10. An engineered Factor XIII barrel protein comprising a Factor XIII β2 barrel protein domain comprising:
seven scaffold regions, and
six intervening regions,
wherein said seven scaffold regions consist of peptides having the sequences of SEQ ID NOS: 658-664, respectively, and at least one of said intervening regions is mutated to have a loop-diversified region having less than 30% sequence identity to SEQ ID NO: 595, and wherein the protein has a beta barrel structure to increase thermostability to provide a melting temperature (Tm) of at least 55° C.