# Patent

## Title
Use of tetracysteine tags in fluorescence-activated cell sorting analysis of prokaryotic cells producing peptides or proteins

## Abstract

A process of in vivo labeling and identifying recombinantly produced peptides or proteins within an unpermeabilized prokaryotic host cell. Recombinant prokaryotic cells expressing a fusion peptide comprising at least one tetracysteine tag were labeled in vivo using a biarsenical labeling reagent. A fluorescent activated cell sorter was used to identify and select subpopulations of fluorescent cells wherein the amount of fusion peptide in the cell was proportional to the amount of fluorescence detected.

## Background
CROSS REFERENCE TO RELATED APPLICATIONS
This application claims the benefit of U.S. Provisional Patent Application No. 60/984,876, filed Nov. 2, 2007.


FIELD OF THE INVENTION
The invention relates to the field of in vivo detection of recombinantly produced peptides or proteins in prokaryotic microbial host cells. More specifically prokaryotic cells that express fusion peptides that comprise at least one tetracysteine tag are labeled in vivo with a biarsenical labeling reagent that binds to the tetracysteine tag and forms a detectable fluorescent complex. These cells are detected or sorted and detected using a fluorescence activated cell sorter [FACS]. The process does not include permeabilizing or pretreating the cells for effective labeling.
BACKGROUND OF THE INVENTION
Efficient production of bioactive proteins and peptides is a primary function of the biomedical and industrial biochemical industry. Bioactive peptides and proteins are used as curative agents in a variety of diseases such as diabetes (insulin), viral infections and leukemia (interferon), diseases of the immune system (interleukins), and red blood cell deficiencies (erythropoietin) to name a few. Additionally, large quantities of proteins and peptides are needed for various industrial applications including, for example, the pulp and paper industries, textiles, food industries, personal care and cosmetics industries, sugar refining, wastewater treatment, production of alcoholic beverages and as catalysts for the generation of new pharmaceuticals.
With the discovery and implementation of combinatorial peptide screening technologies new applications for small peptides having specific binding affinities have been developed. These technologies include bacterial display (Kemp, D. J.; Proc. Natl. Acad. Sci. USA 78(7): 4520-4524 (1981); yeast display (Chien et al., Proc Natl Acad Sci USA 88(21): 9578-82 (1991)), combinatorial solid phase peptide synthesis (U.S. Pat. No. 5,449,754; U.S. Pat. No. 5,480,971; U.S. Pat. No. 5,585,275 and U.S. Pat. No. 5,639,603), phage display technology (U.S. Pat. No. 5,223,409; U.S. Pat. No. 5,403,484; U.S. Pat. No. 5,571,698; and U.S. Pat. No. 5,837,500), ribosome display (U.S. Pat. No. 5,643,768; U.S. Pat. No. 5,658,754; and U.S. Pat. No. 7,074,557), and mRNA display technology (PROFUSIONTM; U.S. Pat. No. 6,258,558; U.S. Pat. No. 6,518,018; U.S. Pat. No. 6,281,344; U.S. Pat. No. 6,214,553; U.S. Pat. No. 6,261,804; U.S. Pat. No. 6,207,446; U.S. Pat. No. 6,846,655; U.S. Pat. No. 6,312,927; U.S. Pat. No. 6,602,685; U.S. Pat. No. 6,416,950; U.S. Pat. No. 6,429,300; U.S. Pat. No. 7,078,197; and U.S. Pat. No. 6,436,665)]
In particular, in biomedical fields small peptides are regarded as linkers for the attachment of diagnostic and pharmaceutical agents to surfaces (see U.S. Pat. App. Pub. No. 2003/0185870 to Grinstaff et al., and U.S. Pat. No. 6,620,419 to Linter), as well as in the personal care industry for the attachment of benefit agents to body surfaces such as hair and skin (see commonly-owned U.S. Pat. No. 7,220,405 to Huang et al., and U.S. Pat. App. Pub. No. 2003/0152976 to Janssen et al.), and in the printing industry for the attachment of pigments to print media (see commonly-owned U.S. Pat. App. Pub. No. 2005/0054752).
Some commercially useful peptides may be synthetically generated or isolated from natural sources. However, these methods are often expensive, time consuming and characterized by limited production capacity. The preferred method of peptide production is through the fermentation of recombinant microorganisms engineered to express the protein or peptide of interest. Although preferable to synthesis or isolation, recombinant peptide production has a number of obstacles to be overcome in order to be cost-effective. For example, peptides and in particular short peptides produced in a cellular environment are susceptible to degradation by native proteases in the cell. Additionally, the purification of some peptides may be difficult depending on the nature of the protein or peptide of interest and may result in poor yields.
One means to mitigate the difficulties associated with recombinant peptide production is the use of chimeric genetic constructs encoding chimeric proteins. The chimeric proteins may comprise at least one portion of the desired protein product fused to at least one portion comprising a peptide tag, referred to herein as “fusion proteins”. The peptide tag may be used to assist protein folding, post expression purification and/or protein passage through the cell membrane and to protect the protein from the action of degradative enzymes,
In many cases it is useful to express a peptide in insoluble form, particularly when the peptide of interest (POI) is a small peptide that is typically soluble under normal physiological conditions and/or subject to endogenous proteolytic degradation within the host cell. Production of the peptide in an insoluble form both facilitates simple recovery and protects the peptide from undesirable proteolytic degradation. One means to produce the peptide of interest in an insoluble form is to recombinantly produce the peptide as part of an insoluble fusion peptide by including at least one peptide tag (referred to herein as a “solubility tag” or “inclusion body tag”) that induces inclusion body formation. The fusion protein may include at least one cleavable peptide linker so that the peptide of interest can be subsequently recovered from the fusion protein. The fusion protein may include a plurality of inclusion body tags, cleavable peptide linkers, and regions comprising the peptide of interest.
Recombinant microbial peptide production often requires the ability to efficiently label, detect/monitor, and/or screen/select cells producing the desired fusion peptide. This ability is useful during both the strain development phase (i.e., identity strains/mutants/growth conditions that improve peptide production) and commercial production phase (i.e. process monitoring). During strain development, it is particularly desirable to identify and select strains exhibiting improved performance using a technique that is sensitive, fast, easy, and non-toxic to the recombinant cell, i.e., permits selection and subsequent growth of the selected cells, and amenable to high-throughput processing or screening.
Various fluorescent labeling and detection techniques have been reported in the art to monitor and/or measure peptide production, although many of these techniques are not cost-effective or suitable for in vivo labeling and detection, especially when producing small peptides. Giepmans et al. (Science 312:217-224 (2006)) reviews the fluorescent “toolbox” for assessing protein production/location and function. Many of the labeling techniques require the use of a targeting molecule to achieve specific labeling, e.g. fusion of small organic dyes and/or quantum dots to antibodies. However, such immunological techniques often require fixation and/or permeabilization and are not amenable to in vivo labeling, especially when one wants to select and grow the cells exhibiting an improvement in peptide production.
Another peptide labeling approach is the incorporation of a detectable fluorescent marker as part of the fusion construct. For example, fluorescent proteins such as green fluorescent protein (GFP) and yellow fluorescent protein (YFP) are often used to detect and/or measure recombinant peptide production. However, fusion constructs comprising a large fluorescent protein are time consuming because they require a significant fluorescence development period) and may place an additional metabolic burden on the microbial host cell. Fusion of a large fluorescent protein to the peptide of interest adversely affects the production efficiency of the peptide of interest, especially when the peptide of interest is small relative to the fluorescent protein. It is desirable to use a detectable marker that is small, easily detectable (sensitive with low background noise), and suitable for in vivo labeling and detection applications. In particular, a simple and effective in vivo labeling system that can be used in combination with a fluorescence activated cell sorter (FACS) for detection and/or selection is desirable.
The LUMIOTM protein detection system (Invitrogen Life Technologies, Carlsbad, Calif.) is based on the incorporation of a small tetracysteine tag (TC) that covalently binds to a biarsenical labeling reagent (e.g. FlAsH-EDT2 [LUMIOTM green]; ReAsh-EDT2 [LUMIOTM red]); and CHoXAsh-EDT2 (U.S. Pat. No. 5,932,474; U.S. Pat. No. 6,054,271; U.S. Pat. No. 6,831,160; U.S. Pat. No. 6,008,378; U.S. Pat. No. 6,451,564; U.S. Pat. No. 6,686,458; U.S. Pat. No. 7,138,503; EP1032837, EP1684073, U.S. Pat. App. Pub. No. 20050176065 A1; and Griffin et al., Science 281:269-271 (1998)). Covalent binding of the labeling reagent to the tetracysteine tag generates a highly fluorescent complex. The LUMIOTM detection system has been extensively used to fluorescently label eukaryotic proteins in vivo, especially mammalian cells and mammalian cell lines (Ho and Starnbach, Infect. Immunity, 73(2):905-911 (2005); Adams et al., JACS, 124:6063-6076 (2002); Stroffekova and Proenza, Eur. J. Physiol., 442:859-866 (2001); Rice et al., Nat. Biotechnol., 19:321-326 (2001); and Int'l App. Pub. No. WO2007/023184A1.
Griffin et al. (Meth. Enzymol., 327:565-578 (2000)) reports that labeling of intact bacterial cells requires much higher concentrations of the biarsenical labeling reagent in the presence of β-mercaptoethanol (2-ME) for several hours (unpublished data), suggesting that the labeling reagent cannot easily penetrate into prokaryotic cells.
Ignatova and Gierash (PNAS, 101 (2):523-528 (2004)) reports in vivo labeling of E. coli cells using a tetracysteine tag/biarsenical labeling reagent system wherein the fluorescence spectra was measured using a fluorometer. However, the labeling process required lysozyme pretreatment to make the outer membrane permeable to the labeling reagent.
A simple and cost effective process for in vivo labeling and detecting TC-tagged proteins produced within prokaryotic cells that does not require the use of undesirable compounds, e.g. β-mercaptoethanol, and/or a permeabilizing pretreatment, e.g. lysozyme treatment, has not been reported. In general, the use of permeabilizing agents and/or reducing agents is undesirable as the treated cells may be non-viable and/or undergo an unpredictable stress response that may influence peptide production and/or the cell's growth characteristics. This is particularly important when the goal of the labeling process is to identify and select viable cells suitable for use in further experiments.
Furthermore, many commercial applications for small bioactive peptides often require purified product. Many of these small peptides are produced in a recombinant prokaryotic host cell in the form of insoluble fusion peptides. A labeling system that is both effective for monitoring fusion peptide production and enables separation from the peptide of interest during subsequent bulk processing is needed.
The problem to be solved is to provide a process of in vivo labeling fusion peptides recombinantly produced within a prokaryotic cell, which is fast, efficient, sensitive, and does not require the use of permeabilized cells. That is, the process does not include the need to contact the cells with an undesirable agent to increase permeability in order to achieve effective in vivo labeling. Furthermore, the process should include a fast and effective means for detecting and/or selecting viable, labeled cells characterized by improved peptide production, e.g. the use of a fluorescence activated cell sorter to collect live cells. In one aspect, the process should be capable of selecting and isolating live cells suitable for use in further experiments and/or selections. In another aspect, the process may include repeatable steps (growth-labeling-detection-selection) that may include at least one round of mutagenesis to facilitate host cell optimization (e.g. increased peptide production).
SUMMARY OF THE INVENTION
The stated problem has been solved through the discovery that peptides or proteins comprising a tetracysteine tag (TC) can be labeled in vivo with biarsenical labeling reagents using prokaryotic host cells that have not been pre-treated with permeabilizing agents and/or reducing agents. The in vivo labeling may be combined with a fluorescence activated cell sorter (FACS), enabling fast and efficient detection and selection of prokaryotic cells producing the desired peptide or protein.
The tetracysteine-tagged peptide or protein may be an insoluble fusion peptide comprising at least one inclusion body tag. The tetracysteine tag may be operably-linked to the portion of the fusion protein comprising the inclusion body tag.
Described herein is process of in vivo labeling and detecting a polypeptide or protein in a prokaryotic cell comprising:
a) providing an unpermeabilized prokaryotic host cell comprising a chimeric genetic construct encoding a polypeptide or protein comprising at least one tetracysteine tag;
b) growing the unpermeabilized prokaryotic host cell of (a) under suitable conditions express the chimeric genetic construct and to produce the polypeptide or protein comprising said at least one tetracysteine tag;
c) contacting in vivo the unpermeabilized prokaryotic host cell of (b) with an effective amount of at least one biarsenical labeling reagent which binds to the at least one tetracysteine tag forming at least one fluorescent complex; and
d) detecting unpermeabilized prokaryotic cells comprising the at least one fluorescent complex.
The chimeric genetic construct may encode a fusion peptide comprising:
i) at least one first portion comprising an inclusion body tag (IBT);
ii) at least one second portion comprising a peptide of interest (POI); and
iii) at least one tetracysteine tag (TC).
The fusion peptide may have the general structure:

IBT-TC-CL-POI or POI-CL-TC-IBT

wherein

    a) CL is a cleavable peptide linker;
    b) the tetracysteine tag comprises amino acid sequence SEQ ID NO: 2;
    c) the biarsenical labeling reagent is selected from the group consisting of fluorescein arsenical hairpin binding reagent and resorufin arsenical hairpin binding reagent; and
    d) the prokaryotic cell is Escherichia coli. 


The affinity peptide is preferably a combinatorially generated peptide identified using phage display, bacterial display, yeast display, ribosome display, or mRNA-display.
The peptide of interest is selected from the group consisting of hair-binding peptides, nail-binding peptides, skin-binding peptides, tooth-binding peptides, antimicrobial peptides, pigment binding peptides, cellulose-binding peptides, polymer-binding peptides, and clay-binding peptides.
The peptide of interest may be a multi-block peptide having a two or more binding moieties wherein the binding moieties may be the same or different.



BRIEF DESCRIPTION OF THE FIGURES
FIGS. 1A-D are plasmid maps of various expression plasmids containing the tetracysteine tag. FIG. 1E illustrates the chimeric genetic construct encoding the peptide of interest targeted for expression as inclusion bodies. The tetracysteine tag (CCPGCC; SEQ ID NO: 2) was cloned between the inclusion body promoting tag (IBT) and the peptide of interest (POI) and upstream of the acid cleavage site, an acid labile aspartic acid-proline (DP) dipeptide.
FIGS. 2A-2F are fluorescence activated cell sorter (FACS) fluorescence intensity plots illustrating increased LUMIOTM in-cell labeling at various induction times. The increase in fluorescence intensity reflects increased peptide production. QC1100 cells expressing the fusion peptide IBT139-CCPGCC-HC776124 with a tetracysteine tag were grown from fresh overnight cultures to OD600 0.5 and induced with 0.2% L-arabinose for different lengths of time. Aliquots of the induced cultures were used for in-cell labeling for FACS analysis (FIGS. 2A-2F) and in-gel LUMIOTM Green labeling (FIG. 2G). The same gel stained with SimplyBlueTM is shown in FIG. 2H). The column labels refer to a molecular weight marker lane (M) and the various induction times (in hours) prior to labeling.
FIGS. 3A and 3B are fluorescence activated cell sorter (FACS) fluorescence intensity plots of the LUMIOTM in-cell labeling of QC1100 cells expressing the fusion peptide IBT139-CCPGCC-HC776124 with the tetracysteine tag. FIG. 3A is a fluorescence intensity plot of cells grown in DEK medium induced at OD600 1.5. FIG. 3B is a fluorescence intensity plot of cells grown in LB medium induced at OD600 0.5. The DEK grown cells or the LB grown cells were induced for three hours with 0.2% L-arabinose and labeled for 1.5 hours as described in Example 7.



BRIEF DESCRIPTION OF THE BIOLOGICAL DEPOSIT
The following biological deposit has been made under the terms of the Budapest Treaty on the International Recognition of the Deposit of Microorganisms for the purposes of Patent Procedure:















Depositor Identification
Int'l. Depository




Reference
Designation
Date of Deposit









Plasmid pCP20
ATCC PTA-4455
Jun. 13, 2002










As used herein, “ATCC” refers to the American Type Culture Collection International Depository Authority located at ATCC, 10801 University Blvd., Manassas, Va. 20110-2209, USA. The “International Depository Designation” is the accession number to the culture on deposit with ATCC.
The listed deposits will be maintained in the indicated international depository for at least thirty (30) years and will be made available to the public upon the grant of a patent disclosing it. The availability of a deposit does not constitute a license to practice the subject invention in derogation of patent rights granted by government action
BRIEF DESCRIPTION OF THE BIOLOGICAL SEQUENCES
The following sequences comply with 37 C.F.R. 1.821-1.825 (“Requirements for Patent Applications Containing Nucleotide Sequences and/or Amino Acid Sequence Disclosures—the Sequence Rules”) and are consistent with World Intellectual Property Organization (WIPO) Standard ST.25 (1998) and the sequence listing requirements of the EPC and PCT (Rules 5.2 and 49.5(a-bis), and Section 208 and Annex C of the Administrative Instructions). The symbols and format used for nucleotide and amino acid sequence data comply with the rules set forth in 37 C.F.R. §1.822.
SEQ ID NO: 1 is the amino acid sequence of the tetracysteine tag CCXXCC.
SEQ ID NO: 2 is the amino acid sequence of the tetracysteine tag CCPGCC.
SEQ ID NOs: 3 and 4 are primers.
SEQ ID NO: 5 is the amino acid sequence of peptide AO9.
SEQ ID NO: 6 is the amino acid sequence of peptide KF11.
SEQ ID NO: 7 is the nucleic acid sequence encoding the peptide HC77643.
SEQ ID NO: 8 is the amino acid sequence of HC77643 (a multi-block hair-binding peptide)
SEQ ID NO: 9 is the nucleic acid sequence encoding the peptide HC776124.
SEQ ID NO: 10 is the amino acid sequence of HC776124 (a multi-block hair-binding peptide).
SEQ ID NO: 11 is the nucleic acid sequence of inclusion body tag IBT139.
SEQ ID NO: 12 is the amino acid sequence of inclusion body tag IBT139.
SEQ ID NO: 13 is the nucleic acid sequence of inclusion body tag IBT139.CCPGCC.
SEQ ID NO: 14 is the amino acid sequence of inclusion body tag IBT139.CCPGCC.
SEQ ID NO: 15 is the nucleic acid sequence encoding inclusion body tag KSI(C4).
SEQ ID NO: 16 is the amino acid sequence of inclusion body tag KSI(C4).
SEQ ID NO: 17 is the amino acid sequence of the core sequence found in a family of inclusion body tags (see co-pending and commonly owned U.S. patent application Ser. No. 11/782,836).
SEQ ID NO: 18 is the nucleic acid sequence of expression plasmid pLR186.
SEQ ID NO: 19 is the nucleic acid sequence of expression plasmid pTG28.
SEQ ID NO: 20 is the nucleic acid sequence of expression plasmid pTG34.
SEQ ID NO: 21 is the nucleic acid sequence of expression plasmid pLR173.
SEQ ID NO: 22 is the nucleic acid sequence of expression plasmid pLR199.
SEQ ID NO: 23 is the nucleic acid sequence of expression plasmid pDCQ500.
SEQ ID NO: 24 is the nucleic acid sequence of expression plasmid pDCQ502.
SEQ ID NO: 25 is the nucleic acid sequence of expression plasmid pDCQ506.
SEQ ID NOs: 8, 10, and 26-49 are the amino acid sequences of hair-binding peptides.
SEQ ID NOs: 50-62 are the amino acid sequences of skin-binding peptides.
SEQ ID NOs: 63-64 are the amino acid sequences of nail-binding peptides.
SEQ ID NOs: 65-93 are the amino acid sequences of antimicrobial peptides.
SEQ ID NOs: 94-119 are the amino acid sequences of pigment-binding peptides. Specifically, SEQ ID NOs: 94-98 bind to carbon black, SEQ ID NOs: 99-106 bind to CROMOPHTAL® yellow (Ciba Specialty Chemicals, Basel, Switzerland), SEQ ID NOs: 107-109 bind to SUNFAST® magenta (Sun Chemical Corp., Parsippany, N.J.), and SEQ ID NOs: 110-119 bind to SUNFAST® blue.
SEQ ID NOs: 120-125 are the amino acid sequences of cellulose-binding peptides.
SEQ ID NOs: 126-153 are the amino acid sequences of polymer-binding peptides. Specifically, SEQ ID NO: 126 binds to poly(ethylene terephthalate), SEQ ID NOs: 127-138 bind to poly(methyl methacrylate), SEQ ID NOs: 139-144 bind to Nylon, and SEQ ID NOs: 145-153 bind to poly(tetrafluoroethylene).
SEQ ID NOs: 154-169 are the amino acid sequences of clay binding peptides.
SEQ ID NO: 170 is the amino acid sequence of the Caspase-3 cleavage sequence.
SEQ ID NOs: 171-219 are the amino acid sequences of various additional inclusion body tags (see co-pending and commonly owned U.S. patent application Ser. Nos. 11/641,936; 11/641,273; 11/516,362; and 12/172,385).
DETAILED DESCRIPTION
Described herein is a process of in vivo labeling and detecting a polypeptide or protein comprising a tetracysteine tag (TC) in a recombinant prokaryotic host cell using a biarsenical labeling reagent. The process does not pretreat the recombinant prokaryotic cells with lysozyme or harsh chemical agents, such as β-mercaptoethanol or toluene prior to in vivo labeling.
The tetracysteine-tagged polypeptide may be recombinantly produced in the prokaryotic host cell in an insoluble form, such as inclusion bodies, by incorporating a solubility tag to form an insoluble fusion protein. Covalent binding of the labeling reagent to the TC tag forms a fluorescent complex that can measure the level fusion peptide production. A fluorescence activated cell sorter (FACS) may be used to identify, measure and/or isolate subpopulations of the prokaryotic cells based on fluorescence intensity.
The prokaryotic host cell may be an enteric bacterial host cell. The enteric bacterial host cell may be Escherichia coli cell. The Escherichia coli cell preferably comprises a disruption in the endogenous arabinose operon (ΔaraBAD) for efficient expression of the fusion peptide under the pBAD promoter.
The following definitions are used herein and should be referred to for interpretation of the claims and the specification.
As used herein, the indefinite articles “a” and “an” preceding an element or component of the invention are intended to be nonrestrictive regarding the number of instances (i.e., occurrences) of the element or component. Therefore “a” or “an” should be read to include one or at least one, and the singular word form of the element or component also includes the plural unless the number is obviously meant to be singular.
As used herein, the term “comprising” means the presence of the stated features, integers, steps, or components as referred to in the claims, but that it does not preclude the presence or addition of one or more other features, integers, steps, components or groups thereof.
As used herein, the term “about” refers to modifying the quantity of an ingredient or reactant of the invention or employed refers to variation in the numerical quantity that can occur, for example, through typical measuring and liquid handling procedures used for making concentrates or use solutions in the real world; inadvertent error in these procedures; differences in the manufacture, source, or purity of the ingredients employed to make the compositions or carry out the methods; and the like. The term “about” also encompasses amounts that differ due to different equilibrium conditions for a composition resulting from a particular initial mixture. Whether or not modified by the term “about”, the claims include equivalents to the quantities.
As used herein, the terms “unpermeabilized prokaryotic host cell”, “unpermeabilized prokaryotic cell”, and “unpermeabilized cell” refer to a prokaryotic cell used in the present process that has not been subjected to a pretreatment prior to labeling the cell in vivo. As used herein, the “pretreatment” will include any agents, such as lysozyme, toluene, used to increase cell wall permeability to the biarsenical labeling reagent(s) or undesirable reducing agents such as β-mercaptoethanol. “Pretreatment” refers to any treatment that occurs prior to, or simultaneously with, contacting the cell in vivo with the biarsenical labeling reagent. Preferably, the present labeling process does not include contacting the prokaryotic cells with lysozyme and/or β-mercaptoethanol any time during the present process.
As used herein, the term “biarsenical labeling reagent” refers to the biarsenical compounds FlAsH-EDT2 [LUMIOTM Green] and ReAsH-EDT2 [LUMIOTM red], CHoXAsh-EDT2 (a blue biarsenical dye; Adams et al., supra) and derivatives thereof that fluoresce when bound to a tetracysteine tag.
As used herein, the terms “FlAsH-EDT2” and “fluorescein arsenical hairpin binding reagent” refer to the compound 4′,5′-bis(1,3,2-dithioarsolan-2-yl)fluorescein-(1,2-ethanedithiol)2. The labeling reagent covalently binds to thiol groups with the tetracysteine tag, generating a fluorescent complex. The excitation maxima has been reported to be 508 nm with an emission maxima at 528 nm.
As used herein, the terms “ReAsH-EDT2” and “resorufin arsenical hairpin binding reagent” refer to 4,5-bis(1,3,2-dithioarsolan-2-yl)-resorufin (complexed with 1,2-ethanedithiol). The labeling reagent covalently binds to thiol groups with the tetracysteine tag, generating a fluorescent complex. The excitation maxima has been reported to be 593 nm with an emission maxima at 608 nm.
As used herein, the term “CHoXAsh-EDT2” refers to the blue biarsenical dye 4,5-bis(1,3,2-dithioarsolan-2-yl)-2,8-dichloro-3,6-dihydroxy-9H-xanthen-9-one (complexed with 1,2-ethanedithiol) (Adams et al., supra).
As used herein, the terms “tetracysteine tag”, “tetracysteine motif”, “LUMIOTM tag”, and “labeling tag” are abbreviated as “TC” and refer to a tetracysteine motif having the formula Cys-Cys-Xaa1-Xaa2-Cys-Cys (SEQ ID NO: 1) wherein Xaa1 and Xaa2 are any naturally occurring amino acid other than cysteine. Derivatives of the tetracysteine motif have been reported to bind the labeling reagents to varying degrees (Adams et al., supra). The tetracysteine tag sequence may preferably be Cys-Cys-Pro-Gly-Cys-Cys (SEQ ID NO: 2). As used herein, a peptide/protein comprising a tetracysteine tag refers to a “TC-tagged” peptide or protein.
As used herein, the term “isolated nucleic acid molecule” is a polymer of RNA or DNA that is single- or double-stranded, optionally containing synthetic, non-natural or altered nucleotide bases. An isolated nucleic acid molecule in the form of a polymer of DNA may be comprised of one or more segments of cDNA, genomic DNA or synthetic DNA.
As used herein, the term “pigment” refers to an insoluble, organic or inorganic colorant.
As used herein, the term “hair” as used herein refers to human hair, eyebrows, and eyelashes.
As used herein, the term “skin” refers to human skin, or substitutes for human skin, such as pig skin, VITRO-SKIN® and EPIDERMTM. Skin, as used herein, will refer to a body surface generally comprising a layer of epithelial cells and may additionally comprise a layer of endothelial cells.
As used herein, the term “nails” as used herein refers to human fingernails and toenails.
As used herein, “PBP” is an abbreviation for polymer-binding peptide. As used herein, the term “polymer-binding peptide” refers to peptide sequences that bind with high affinity to a specific polymer (U.S. patent application Ser. No. 11/516,362). Examples include peptides that bind to poly(ethylene terephthalate) (SEQ ID NO: 126), poly(methyl methacrylate) (SEQ ID NOs: 127-138), Nylon (SEQ ID NOs: 139-144), and poly(tetrafluoroethylene) (SEQ ID NOs: 145-153).
As used herein, “HBP” is an abbreviation for hair-binding peptide. As used herein, the term “hair-binding peptide” refers to peptide sequences that bind with high affinity to hair. Examples of hair binding peptides have been reported (U.S. patent application Ser. No. 11/074,473 to Huang et al.; Int'l. App. Pub. No. WO 0179479; U.S. Pat. App. Pub. No. 2002/0098524 to Murray et al.; Janssen et al., U.S. Pat. App. Pub. No. 2003/0152976 to Janssen et al.; Int'l App. Pub. No. WO 2004048399; U.S. patent application Ser. No. 11/512,910, and U.S. Pat. App, No. 11/696,380). Hair-binding peptides may include one or more hair binding domains. As used herein, hair-binding peptides comprising of a plurality of hair-binding domains are referred to herein as “multi-block” or “multi-copy” hair-binding peptides. Examples of hair-binding peptides are provided as SEQ ID NOs: 8, 10, and 26-49.
As used herein, “SBP” is an abbreviation for skin-binding peptide. As used herein, the term “skin-binding peptide” refers to peptide sequences that bind with high affinity to skin. Examples of skin-binding peptides have also been reported (U.S. patent application Ser. No. 11/069,858 to Buseman-Williams; Int'l App. Pub. No. WO 2004/000257 to Rothe et al.; and U.S. patent application Ser. No. 11/696,380). Skin is a body surface comprises a layer of epithelial cells and may additionally comprise a layer of endothelial cells. Examples of skin-binding peptides are provided as SEQ ID NOs: 50-62.
As used herein, “NBP” is an abbreviation for nail-binding peptide. As used herein, the term “nail-binding peptide” refers to peptide sequences that bind with high affinity to nail. Nail-binding peptides have been reported (U.S. patent application Ser. No. 11/696,380). Examples of nail-binding peptides are provided as SEQ ID NOs: 63-64.
As used herein, an “antimicrobial peptide” is a peptide having the ability to kill microbial cell populations (U.S. patent application Ser. No. 11/516,362). Examples of antimicrobial peptides are provided as SEQ ID NOs: 65-93.
As used herein, “cellulose-binding peptide” refers to a peptide that binds with high affinity to cellulose. Examples of cellulose-binding peptides are provided as SEQ ID NOs: 120-125.
As used herein, “clay-binding peptide” refers to a peptide that binds with high affinity to clay (U.S. patent application Ser. No. 11/696,380). Examples of clay-binding peptides are provided as SEQ ID NOs: 154-169.
As used herein, “multi-block peptides” refers to a peptide comprising at least two binding moieties. Each binding moiety has an affinity for a target substrate (e.g. hair, skin, a pigment, etc). The binding moieties may have an affinity for the same or different substrates (for example, a hair-binding moiety fused to a pigment binding moiety for targeted delivery of a pigment to hair or a peptide having a plurality of hair-binding moieties). The construction of multi-block hair-binding peptides has been reported (see co-pending and commonly owned U.S. patent application Ser. Nos. 11/389,948 and 11/074,473).
As used herein, the term “inclusion body tag” is abbreviated “IBT” and refers to a polypeptide that facilitates formation of inclusion bodies when fused to a peptide of interest. The peptide of interest is typically soluble within the host cell and/or host cell lysate when not fused to an inclusion body tag. Fusion of the peptide of interest to the inclusion body tag produces a fusion protein that agglomerates into intracellular bodies (inclusion bodies) within the host cell.
As used herein, “cleavable linker elements”, “peptide linkers”, “cleavable peptide linkers”, and “cleavage site” will be used interchangeably and refer to cleavable peptide segments located between the inclusion body tag and the peptide of interest or between the tetracysteine tag and the peptide of interest. In a preferred embodiment, the portion of the fusion protein comprising the inclusion body tag further comprises at least one tetracysteine tag. After the inclusion bodies are separated, partially-purified or purified from the cell lysate (typically reserved for bulk processing), the linker elements can be cleaved chemically and/or enzymatically to separate the inclusion body tag from the peptide of interest. The fusion peptide may also include a plurality of regions encoding one or more peptides of interest separated by one or more cleavable peptide linkers. The peptide of interest can then be isolated from the inclusion body tag, if necessary. It should be noted that isolation of POI is typically reserved for bulk processing of fermented cells and not the sample aliquots used for the present labeling and detection process. The inclusion body tag(s) and the peptide of interest may have different solubilities in a defined medium, such as aqueous solution. The solubility difference enables separation of the inclusion body tag from the polypeptide of interest. The inclusion body tag may preferably be insoluble in an aqueous solution while the protein/polypeptide of interest is appreciably soluble in an aqueous solution. The pH, temperature, and/or ionic strength of the aqueous solution can be adjusted to facilitate recovery of the peptide of interest. The difference in solubility between the inclusion body tag and the peptide of interest may preferably occur in an aqueous solution having a pH of 5 to 10 and a temperature range of about 15° C. to about 50° C.
The cleavable peptide linker may be from 1 to about 50 amino acids, preferably from 1 to about 20 amino acids in length, and most preferably about 2 to about 10 amino acids in length. An example of an enzymatically cleavable peptide linker is provided by SEQ ID NO: 170 (Caspase-3 cleavage recognition sequence). Preferably, the cleavage site is an acid cleavable aspartic acid-proline dipeptide (D-P) moiety. The cleavable peptide linkers may be incorporated into the fusion proteins using any number of techniques well known in the art. The inclusion body tag may also comprise an effective number of cross-linkable cysteine residues whereby oxidative cross-linking can be used to selectively precipitate the IBT once cleaved from the POI (see co-pending and commonly owned U.S. patent application Ser. No. 12/172,395).
As used herein, the term “operably-linked” refers to the association of nucleic acid sequences on a single nucleic acid fragment so that the function of one is affected by the other. For example, a promoter is operably-linked with a coding sequence when it is capable of affecting the expression of that coding sequence, that is, that the coding sequence is under the transcriptional control of the promoter. The definition of “operably linked” may also be extended to describe the products of chimeric genes, such as fusion peptides. As such, “operably-linked” refers to the linking of an inclusion body tag to a peptide of interest to be produced and recovered.
As used herein, the terms “fusion peptide”, “fusion protein”, “chimeric protein”, and “chimeric peptide” are used interchangeably and refer to a polymer of amino acids—peptide, oligopeptide, polypeptide, or protein—comprising at least two portions, each portion comprising a distinct function. At least one first portion of the fusion peptide comprises at least one inclusion body tag. At least one second portion of the fusion peptide comprises at least one peptide of interest. The fusion peptide comprises at least one tetracysteine tag. The tetracysteine tag may be separated from the peptide of interest using a cleavable peptide linker. The inclusion body tag may comprise the tetracysteine tag wherein the peptide of interest targeted for use in commercial product formulations does not include the tetracysteine tag. The tetracysteine tag may be located on the amino and/or carboxy terminus of the inclusion body tag and is separated from the peptide of interest by at least one cleavable peptide linker.
As used herein, the terms “polypeptide” and “peptide” are used interchangeably to refer to a polymer of two or more amino acids joined together by a peptide bond, wherein the peptide is of unspecified length, thus, peptides, oligopeptides, polypeptides, and proteins are included within the present definition. This term also includes post expression modifications of the polypeptide, for example, glycosylations, acetylations, phosphorylations and the like. Included within the definition are, for example, peptides containing one or more analogues of an amino acid or labeled amino acids and peptidomimetics. Preferably, the IBTs are comprised of L-amino acids.
As used herein, the terms “protein of interest”, “polypeptide of interest”, “peptide of interest”, “targeted protein”, “targeted polypeptide”, “targeted peptide”, “expressible protein of interest”, and “expressible polypeptide of interest” are used interchangeably to refer to a protein, polypeptide, or peptide that is bioactive and may be expressed by the genetic machinery of a host cell. The peptide of interest may be abbreviated as “POI”. The peptide of interest may be an affinity peptide, i.e., a peptide having a specific affinity for a defined substrate, of no more than 300 amino acids in length, preferably no more than 200 amino acids in length, more preferably no more than 100 amino acids in length, and most preferably no more than 50 amino acids in length. The affinity peptide of interest may be an affinity peptide selected from a combinatorially-generated library wherein the affinity peptide was selected using a display technique selected from the group consisting of phage display, yeast display, ribosome display, and mRNA display.
As used herein, the term “bioactive” or “peptide of interest activity” refers to the activity or characteristic associated with the peptide and/or protein of interest. The bioactive peptides may be used in a variety of applications including, but not limited to curative agents for diseases (e.g., insulin, interferon, interleukins, anti-angiogenic peptides (U.S. Pat. No. 6,815,426), and polypeptides that bind to defined cellular targets with the proviso that the peptide of interest is not an antibody or the Fab fragment of an antibody, such as receptors, channels, lipids, cytosolic proteins, and membrane proteins, peptides having antimicrobial activity, peptides having an affinity for a particular material, such as hair-binding polypeptides, skin-binding polypeptides, nail-binding polypeptides, tooth binding peptides, cellulose binding polypeptides, polymer-binding polypeptides, clay-binding polypeptides, and peptides that have an affinity for particular animal or plant tissues, for targeted delivery of benefit agents. In a preferred embodiment, the peptide of interest binds to a body surface such as skin, hair, nail, or teeth.
As used herein, the “benefit agent” refers to a molecule that imparts a desired functionality to the complex for a defined application. The benefit agent may be peptide of interest itself or may be one or more molecules bound to covalently or non-covalently, or associated with, the peptide of interest wherein the binding affinity of the targeted polypeptide is used to selectively target the benefit agent to the targeted material. The targeted polypeptide may comprise at least one region having an affinity for at least one target material, such as biological molecules, polymers, hair, skin, nail, clays, and other peptides and at least one region having an affinity for the benefit agent, such as pharmaceutical agents, pigments, conditioners, dyes, fragrances, and the like. The peptide of interest may comprise a plurality of regions having an affinity for the target material and a plurality of regions having an affinity for the benefit agent. The peptide of interest may comprise at least one region having an affinity for a targeted material and a plurality of regions having an affinity for a variety of benefit agents wherein the benefit agents may be the same of different. Examples of benefits agents may include, but are not limited to conditioners for personal care products, pigments, dyes, fragrances, pharmaceutical agents (e.g., targeted delivery of cancer treatment agents), diagnostic/labeling agents, ultraviolet light blocking agents (i.e., active agents in sunscreen protectants), and antimicrobial agents (e.g., antimicrobial peptides), to name a few. The benefit agent may be covalently attached to the affinity peptide using any number of chemical cross-linking techniques well-known in the art.
As used herein, the term “inclusion body” refers to an intracellular amorphous deposit comprising aggregated protein found in the cytoplasm of a cell. Peptides of interest that are typically soluble with the host cell and/or cell lysate can be fused to one or more of the present inclusion body tags to facilitate formation of an insoluble fusion protein. Alternatively, the peptide of interest may be partially insoluble in the host cell, but produced at relatively lows levels where significant inclusion body formation does not occur. As such, the formation of inclusion bodies will increase peptide production. Fusion of the peptide of interest to one or more inclusion body tags (IBTs) may be used to increase the amount of protein produced in the host cell. Formation of the inclusion body facilitates simple and efficient purification of the fusion peptide from the cell lysate using techniques well known in the art, such as centrifugation and filtration. The inclusion body tag may also comprise an effective number of cross-linkable cysteine residues useful for separating the IBT from the peptide of interest post cleavage into a mixture of peptide fragments with the proviso that the peptide of interest is devoid of cross-linkable cysteine residues. The fusion protein may include one or more cleavable peptide linkers used to separate the protein/polypeptide of interest from the inclusion body tag(s). The cleavable peptide linker can be cleaved chemically, e.g., acid hydrolysis, and/or enzymatically. A protease/peptidase may be used that preferentially recognizes an amino acid cleavage site and/or sequence within the cleavable peptide linker.
“Codon degeneracy” refers to the nature in the genetic code permitting variation of the nucleotide sequence without affecting the amino acid sequence of an encoded polypeptide. The skilled artisan is well aware of the “codon-bias” exhibited by a specific host cell in usage of nucleotide codons to specify a given amino acid. When synthesizing a gene for improved expression in a host cell it is generally desirable to design the gene such that its frequency of codon usage approaches the frequency of preferred codon usage of the host cell.
As used herein, the term “solubility” refers to the amount of a substance that can be dissolved in a unit volume of a liquid under specified conditions. The term “solubility” may also be used to describe the ability of a peptide—inclusion body tag, peptide of interest, or fusion peptides—to be dissolved in a volume of solvent, such as a biological buffer, under specified conditions. The peptides targeted for production may be substantially soluble in the cell and/or cell lysate under normal physiological conditions. Fusion of one or more inclusion body tags (IBTs) to the target peptide results in the formation of a fusion peptide that is insoluble under normal physiological conditions. The peptide of interest may be soluble in an aqueous matrix having a pH range of 5-12, preferably a pH of 6-10; and a temperature range of 5° C. to 50° C., preferably 10° C. to 40° C.
The term “amino acid” refers to the basic chemical structural unit of a protein or polypeptide. The following abbreviations are used herein to identify specific amino acids:
















Three-Letter
One-Letter



Amino Acid
Abbreviation
Abbreviation









Alanine
Ala
A



Arginine
Arg
R



Asparagine
Asn
N



Aspartic acid
Asp
D



Cysteine
Cys
C



Glutamine
Gln
Q



Glutamic acid
Glu
E



Glycine
Gly
G



Histidine
His
H



Isoleucine
Ile
I



Leucine
Leu
L



Lysine
Lys
K



Methionine
Met
M



Phenylalanine
Phe
F



Proline
Pro
P



Serine
Ser
S



Threonine
Thr
T



Tryptophan
Trp
W



Tyrosine
Tyr
Y



Valine
Val
V



Any amino acid (or as defined
Xaa
X



herein)










As used herein, the term “gene” refers to a nucleic acid fragment that expresses a specific protein, including regulatory sequences preceding (5′ non-coding sequences) and following (3′ non-coding sequences) the coding sequence. The term “native gene” refers to a gene as found in nature with its own regulatory sequences The term “chimeric gene” refers to any gene that is not a native gene, comprising regulatory and/or coding sequences including coding sequences of fusion peptides that are not found together in nature. Accordingly, a chimeric gene may comprise regulatory sequences and coding sequences that are derived from different sources, or regulatory sequences and coding sequences derived from the same source, but arranged in a manner different than that found in nature. A “foreign” gene refers to a gene not normally found in the host organism, but that is introduced into the host organism by gene transfer.
As used herein, the term “genetic construct” refers to a series of contiguous nucleic acids useful for modulating the genotype or phenotype of an organism. Non-limiting examples of genetic constructs include but are not limited to a nucleic acid molecule, and open reading frame, a gene, a plasmid and the like.
As used herein, the term “mutagenic procedure” refers to any procedure or process capable of introducing a genetic alteration or mutation within the host cell and may include any process that results in an addition, deletion, or substitution to the genetic material within the host cell. Examples of mutagenic procedures may include, but are not limited to error-prone PCR, DNA shuffling, site-directed mutagenesis, transposon mutagenesis, recombinant DNA expression, random mutagenesis such as chemical mutagenesis, ultraviolet radiation, ionizing radiation, spontaneous mutations, and the like, promoter replacement, alterations in gene copy number, antisense expression, and down-regulation/disruption of endogenous gene expression, to name a few. The effect of the mutagenic procedure may be measured by the relative level of fluorescence detected. The recombinant prokaryotic cells exhibiting increased fluorescence after being subjected to a mutagenic procedure may be selected as the preferred subpopulation using FACS.
As used herein, the term “host cell” refers to cell that has been transformed or transfected or is capable of being transformed or transfected with an exogenous polynucleotide sequence. The present process is directed to the use of a recombinant prokaryotic host cell to produce a fusion peptide comprising a tetracysteine tag. The recombinant prokaryotic host cell may preferably be a bacteria, preferably an enteric bacteria, and most preferably Escherichia coli. 
As used herein, the term “subpopulation” refers to a portion of the total cells analyzed and selected by the present process. Typically, a “subpopulation” of the cells is selected from the total population of cells analyzed by FACS based on a specified selection criteria measured by the instrument. The subpopulation may be selected based on the measured fluorescence, preferably those cells exhibiting relatively higher fluorescence within the total population of cells. The selected population of cells typically undergoes repeated rounds of growth, expression, labeling/detection, and selection in order to obtain cells with the highest fluorescence. The repeated process further may include at a least one additional step capable of introducing at least one genetic alteration to the host cell using a mutagenic procedure. Additional parameters such as cell size, cell shape, or inclusion body content may also be included (in addition to fluorescence) in the selection criteria.
As used herein, the terms “plasmid”, “vector” and “cassette” refer to an extrachromosomal element often carrying genes which are not part of the central metabolism of the cell, and usually in the form of circular double-stranded DNA molecules. Such elements may be autonomously replicating sequences, genome integrating sequences, phage or nucleotide sequences, linear or circular, of a single- or double-stranded DNA or RNA, derived from any source, in which a number of nucleotide sequences have been joined or recombined into a unique construction which is capable of introducing a promoter fragment and DNA sequence for a selected gene product along with appropriate 3′ untranslated sequence into a cell. “Transformation cassette” refers to a specific vector containing a foreign gene and having elements in addition to the foreign gene that facilitates transformation of a particular host cell. “Expression cassette” refers to a specific vector containing a foreign gene and having elements in addition to the foreign gene that allow for enhanced expression of that gene in a foreign host.
The term “binding affinity” refers to the strength of the interaction of a binding peptide with its respective substrate. The binding affinity can be defined or measured in terms a dissociation constant (KD), and MB50 value. In one embodiment, the binding affinity is determined in terms of the MB50 value using an ELISA-based binding assay.
The term “MB50” refers to the concentration of the binding peptide that gives a signal that is 50% of the maximum signal obtained in an ELISA-based binding assay. The MB50 provides an indication of the strength of the binding interaction or affinity of the components of the complex. The lower the value of MB50, the stronger the interaction of the peptide with its corresponding substrate.
Standard recombinant DNA and molecular cloning techniques used herein are well known in the art and are described by Sambrook, J. and Russell, D., Molecular Cloning: A Laboratory Manual, Third Edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (2001); and by Silhavy, T. J., Bennan, M. L. and Enquist, L. W., Experiments with Gene Fusions, Cold Spring Harbor Laboratory Cold Press Spring Harbor, N.Y. (1984); and by Ausubel, F. M. et. al., Short Protocols in Molecular Biology, 5th Ed. Current Protocols and John Wiley and Sons, Inc., N.Y., 2002.
Inclusion Body Tags
Fusion proteins comprising a carrier protein tag (“inclusion body fusion partner”) that facilitates the expression of insoluble proteins are well-known in the art. The art typically uses inclusion body fusion partners, also referred to herein as “inclusion body tags”, “IBTs” or “solubility tags”, that are quite large, increasing the likelihood that the fusion protein will be insoluble. Example of large peptide tags typically used include, but are not limited to chloramphenicol acetyltransferase (Dykes et al., Eur. J. Biochem., 174:411 (1988), β-galactosidase (Schellenberger et al., Int. J. Peptide Protein Res., 41:326 (1993); Shen et al., Proc. Nat. Acad. Sci. USA 281:4627 (1984); and Kempe et al., Gene, 39:239 (1985)), glutathione-S-transferase (Ray et al., Bio/Technology, 11:64 (1993) and Hancock et al. (Int'l App. Pub. No. WO94/04688)), the N-terminus of L-ribulokinase (U.S. Pat. No. 5,206,154 and Lai et al., Antimicrob. Agents & Chemo., 37:1614 (1993), bacteriophage T4 gp55 protein (Gramm et al., Bio/Technology, 12:1017 (1994), bacterial ketosteroid isomerase (KSI) protein (Kuliopulos et al., J. Am. Chem. Soc. 116:4599 (1994) and U.S. Pat. No. 5,648,244), ubiquitin (Pilon et al., Biotechnol. Prog., 13:374-79 (1997), bovine prochymosin (Haught et al., Biotechnol. Bioengineer. 57:55-61 (1998), and bactericidal/permeability-increasing protein (“BPI”; Better, M. D. and Gavit, P D., U.S. Pat. No. 6,242,219). The art is replete with specific examples of this technology, see for example U.S. Pat. No. 6,613,548, describing fusion protein of proteinaceous tag and a soluble protein and subsequent purification from cell lysate; U.S. Pat. No. 6,037,145, teaching a tag that protects the expressed chimeric protein from a specific protease; U.S. Pat. No. 5,648,244, teaching the synthesis of a fusion protein having a tag and a cleavable linker for facile purification of the desired protein; and U.S. Pat. No. 5,215,896; U.S. Pat. No. 5,302,526; U.S. Pat. No. 5,330,902; and U.S. Pat. App. Pub. No. 2005/221444, describing fusion tags containing amino acid compositions specifically designed to increase insolubility of the chimeric protein or peptide.
Shorter inclusion tags have recently been developed from the Zea mays zein protein (co-pending and commonly owned U.S. Pat. App. No. 11/641,936), the Daucus carota cystatin protein (co-pending and commonly owned U.S. patent application Ser. No. 11/641,273), and an amyloid-like hypothetical protein from Caenorhabditis elegans (co-pending and commonly owned U.S. patent application Ser. No. 11/516,362; SEQ ID NOs: 171-218). The use of short inclusion body tags increases the total amount of the target peptide produced.
A family of small inclusion body tags was recently developed from amyloid-like proteins having amyloid fibrillar morphologies and often exhibit β-sheet tape architecture. An 11 amino acid synthetic peptide (i.e. peptide “PII-2”; also known as peptide “DN1”) capable of self-assembly into β-sheet tapes, ribbons, fibrils, and fibers in water has been described (Aggeli et al., J. Amer. Chem. Soc., 125:9619-9628 (2003); Aggeli et al., PNAS, 98(21):11857-11862 (2001); Aggeli et al., Nature, 386:259-262 (1997); and Aggeli et al., J. Mater Chem, 7(7):1135-1145 (1997). The P11-2 peptide served as the core sequence for the preparation of a family of structurally-related inclusion body tags; including IBT139 and IBT139.CCPGCC (SEQ ID NO:2) (See co-pending and commonly owned U.S. patent application Ser. No. 11/782,836). This family of structurally-related inclusion body tags comprising at least two copies of the core sequence Gln-Gln-Xaa1-Phe-Xaa2-Trp-Xaa3-Phe-Xaa4-Xaa5-Gln (SEQ ID NO: 17), wherein Xaa1=Arg, His, or Lys; Xaa2=Gln, His, or Lys; Xaa3=Gln, His, or Lys; Xaa4=Glu or Gln; and Xaa5=Gln or Lys. IBT139 (SEQ ID NOs: 11 and 12) was used in the present examples to prepare a tetracysteine tagged inclusion body tag (IBT139.CCPGCC; SEQ ID NOs: 13 and 14).
An additional inclusion body tag derived from ketosteroid isomerase (“KSI”; Kuliopulos et al., J. Am. Chem. Soc. 116:4599 (1994) and U.S. Pat. No. 5,648,244) is also used in the present examples. The modified KSI derivative, referred to herein as “KSI(C4)” (SEQ ID NOs: 15 and 16), has been previously reported in co-pending and commonly owned U.S. patent application Ser. Nos. 11/512,910; 11/514,804; and 11/389,948; and U.S. Pat. App. Nos. 60/869,363; 60/855,251; and 60/857,105). The KSI(C4) tag comprises an effective number of cross-linkable cysteine residues that enable separation of the solubility tag from the peptide of interest by oxidative cross-linking (see co-pending and commonly owned U.S. patent application Ser. No. 12/172,395). As illustrated in the present examples, the KSI(C4) inclusion body was modified to include a tetracysteine tag (SEQ ID NO: 2) on the C-terminal end of the tag (FIG. 1A, pDCQ500 (SEQ ID NO: 23)).
An acid-resistance version of the KSI(C4) tag [“KSI(C4)E”] has recently been reported in co-pending and commonly owned U.S. patent application Ser. No. 12/172,385 and is provided as SEQ ID NO: 219.
Expressible Peptides/Proteins of Interest—Large Peptides/Proteins
The process described herein may be used to in vivo label and identify any tetracysteine-tagged peptide, polypeptide or protein recombinantly produced in an unpermeabilized prokaryotic host cell. Construction and expression of suitable genetic constructs is well-known in the art. The genetic construct may be designed so that at least one tetracysteine tag is operably linked to the N- or C-terminus of the peptide/protein and may also be incorporated internally into the peptide/protein of interest. The chimeric genetic construct may be designed so that the tetracysteine tag is operably linked to the N- or C-terminus of the peptide/protein to be expressed and labeled.
Expressible Peptides of Interest—Short Peptides of Interest Produced in the Form of Fusion Peptides
As described herein, the peptide of interest (“expressible peptide”) targeted for production using the present process is one that is substantially soluble in the host cell and/or host cell liquid lysate under normal physiological conditions. The peptide of interest may generally be small, often less than 500 amino acids in length, and difficult to produce in sufficient amounts due to endogenous proteolytic degradation. Fusion of the peptide of interest to at least one solubility tag creates a fusion peptide that is insoluble in the host cell and/or host cell lysate under normal physiological conditions. Production of the peptide of interest is typically increased when expressed and accumulated in the form of an insoluble inclusion body as the peptide is generally more protected from proteolytic degradation. Furthermore, the insoluble fusion protein can be easily separated from the host cell lysate using any number of simple separation techniques, such as centrifugation and/or filtration. The peptide of interest may be produced in the form of a fusion peptide. The fusion peptide may be an insoluble fusion peptide that accumulates in the host cell in the form of inclusion bodies.
The length of the peptide of interest may vary as long as (1) the peptide is appreciably soluble in the host cell and/or cell lysate, and/or (2) the amount of the targeted peptide produced is significantly increased when expressed in the form of an insoluble fusion peptide/inclusion body (i.e. expression in the form of a fusion protein protect the peptide of interest from proteolytic degradation). The peptide of interest can be no more than 500 amino acids in length, preferably no more than 300 amino acids in length, more preferably less than 200 amino acids in length, even more preferably less than 100 amino acids in length, even more preferably less than 75 amino acids in length, yet even more preferably less than 50 amino acids in length, and most preferably less than 30 amino acids in length.
The function of the peptide of interest is not limited by the process described herein and may include, but is not limited to bioactive molecules such as curative agents for diseases (e.g., insulin, interferon, interleukins, peptide hormones, anti-angiogenic peptides, and peptides with the proviso that the peptide is not an antibody or an Fab portion of an antibody that bind to and affect defined cellular targets such as receptors, channels, lipids, cytosolic proteins, and membrane proteins (see U.S. Pat. No. 6,696,089), peptides having an affinity for a particular material, e.g., biological tissues/body surfaces such as hair-binding peptides (U.S. patent application Ser. No. 11/074,473; Int'l Pat. App. No. WO 0179479; U.S. Pat. App. Pub. No. 2002/0098524; U.S. Pat. App. Pub. No. 2003/0152976; Int'l App. Pub. No. WO 04048399; U.S. patent application Ser. No. 11/512,910; U.S. patent application Ser. No. 11/516,362; and U.S. patent application Ser. No. 11/696,380), skin-binding peptides (U.S. patent application Ser. No. 11/069,858; Int'l App. Pub. No. WO 2004/000257; U.S. patent application Ser. No. 11/516,362; and U.S. patent application Ser. No. 11/696,380), nail-binding peptides (U.S. patent application Ser. No. 11/074,473; U.S. patent application Ser. No. 11/696,380)) and peptides having specific affinity for other materials such as cellulose-binding peptides, polymer-binding peptides (U.S. patent application Ser. Nos. 11/607,723,11/607,792, 11/607,734,11/607,672, and 11/607,673), and clay-binding peptides (U.S. patent application Ser. No. 11/696,380), for targeted delivery of at least one benefit agent (see U.S. Pat. No. 7,220,405; U.S. patent application Ser. No. 11/074,473; and U.S. patent application Ser. No. 11/696,380).
The peptide of interest may be a single-stranded affinity peptide derived from a combinatorially-generated library. Preferably, the affinity peptide may be no more than 300 amino acids in length. The affinity peptide may have a binding affinity for a target substrate, as measured by MB50 values, of less than or equal to about 10−5 M, less than or equal to about 10−6 M, less than or equal to about 10−7 M, less than or equal to about 10−8 M, preferably less than or equal to about 10−9 M, and more preferably less than or equal to about 10−10 M.
The affinity peptide may be selected from the group consisting of hair-binding peptides, nail-binding peptides, skin-binding peptides, tooth-binding peptides, pigment binding peptides, cellulose-binding peptides, polymer-binding peptides, and clay-binding peptides.
The peptide of interest may preferably comprise a body surface-binding peptide comprising at least one body surface-binding moiety. The hair-binding peptide may be selected from the group consisting of SEQ ID NOs: 8, 10, and 26-49. The skin-binding peptide may be selected from the group consisting of SEQ ID NOs: 50-62. The nail-binding peptide may be selected from the group consisting of SEQ ID NOs: 63 and 64. The polymer-binding peptide may be selected from the group consisting of SEQ ID NOs: 126-153. The pigment-binding peptide may be selected from the group consisting of SEQ ID NOs: 94-119. The cellulose-binding peptide may be selected from the group consisting of SEQ ID NOs: 120-125. The clay-binding peptide may be selected from the group consisting of SEQ ID NOs: 154-169. The peptide of interest may be a multi-block hair-binding peptide (see U.S. patent application Ser. Nos. 11/074,473 and 11/389,948).
The peptide of interest may have antimicrobial activity. The antimicrobial peptide may be selected from the group consisting of SEQ ID NOs: 65-93.
Multi-block hair binding peptides HC77643 (referred to herein as “HC43”) and HC776124 (also referred to herein as “HC124”) have been described previously. Both peptides are used herein as examples of suitable peptides of interest, although the present process may be used to measure any recombinant prokaryotic cells comprising a TC-tagged peptide. The peptide of interest may be an affinity peptide having a specific affinity for a body surface (e.g. skin, eye, oral, hair, teeth, etc.). The peptide of interest may be an affinity peptide identified by biopanning against a combinatorially-generated library. Preferably the affinity peptide is identified using a biopanning technique selected from the group consisting of phage display, yeast display, ribosome display, and mRNA display.
The two multi-block hair-binding peptides were designed with the following amino acid sequences. Construction of multi-block hair binding peptides have been reported (see co-pending and commonly owned U.S. patent application Ser. Nos. 11/389,948 and 11/074,473). Each of the multi-block hair binding peptides comprises one or more hair binding domains. The functional binding domains are provided in Table 1. Hair-binding domains (bold) include A09 (IPWWNIRAPLNA; SEQ ID NO: 5) and KF11 (NTSQLST; SEQ ID NO: 6). The affinity domains with the multi-block peptides are typically separated by short peptide spacers. The DP acid cleavable moieties are italicized.







TABLE 1







Multi-block affinity peptides used in the present examples.













Formula







(Functional Binding

Nucleic acid
Amino Acid


Peptide Name
Domains in Bold)
Amino acid Sequence
SEQ ID NO:
SEQ ID NO:





HC77643
GSDPG-A09-GAG-
GSDPGIPWWNIRAPLNAGAGIPWWNIRAPLNA
7
8



or “HC43”
A09-GGSGPGSGG-
GGSGPGSGGNTSQLSTGGGNTSQLST



KF11-GGG-KF11-
GGPKK



GGPKK


 


HC776124
GSD(PG-A09-GAG-
GSDPGIPWWNIRAPLNAGAGIPWWNIRAPLNA
9
10


or “HC124”
A09-GGSGPGSGG-
GGSGPGSGGNTSQLSTGGGNTSQLSTGGPK



KF11-GGG-KF11-
KPGDPGIPWWNIRAPLNAGAGIPWWNIRAPLN



GGPKKPGD)2
AGGSGPGSGGNTSQLSTGGGNTSQLSTGGP




KKPGD









Affinity peptides are particularly useful to target benefit agents imparting a desired functionality to a target material (such as hair, skin, nail, or teeth) for a defined application (see U.S. Pat. No. 7,220,405; U.S. patent application Ser. No. 11/074,473; U.S. patent application Ser. No. 11/512,910; and U.S. patent application Ser. No. 11/696,380 for a list of typical benefit agents such as conditioners, pigments/colorants, fragrances, etc.). The benefit agent may be a peptide of interest itself or may be one or more molecules bound to (covalently or non-covalently), or associated with, the peptide of interest wherein the binding affinity of the peptide of interest is used to selectively target the benefit agent to the targeted material. The peptide of interest may comprise at least one region having an affinity for at least one target material, such as, biological molecules, polymers, hair, skin, nail, other peptides, etc., and at least one region having an affinity for the benefit agent, such as, pharmaceutical agents, antimicrobial agents, colorants, dyes, pigments, polymer coated pigments, conditioners, dyes, fragrances, etc. The peptide of interest may comprise a plurality of regions having an affinity for the target material and a plurality of regions having an affinity for one or more benefit agents. The peptide of interest may comprise at least one region having an affinity for a targeted material and a plurality of regions having an affinity for a variety of benefit agents wherein the benefit agents may be the same of different.

Cleavable Peptide Linkers

The use of cleavable peptide linkers, i.e. cleavage sites or cleavage sequences, is well-known in the art. Cleavage peptide linkers are typically incorporated into the peptide/protein construct to facilitate downstream processing. A cleavable peptide linker may be used to separate the tetracysteine tag from the peptide/protein targeted for labeling.
The peptide of interest may also be a fusion peptide. Fusion peptides comprising the inclusion body tags will typically include at least one cleavable peptide sequence separating the inclusion body tag from the polypeptide of interest. The cleavable sequence facilitates separation of the inclusion body tag(s) from the peptide(s) of interest. Cleavage of the peptide linker is typically reserved for bulk processing of the fusion peptides obtained from the fermented cell biomass and should not be used for labeling and detection of the small aliquots of cells from shake flasks or from smaller scale fermentors, such as those described herein. However, the cleavable linker is typically incorporated into the constructs used in the present process. Preferred cells, such as those producing higher amounts of the desired peptide of interest as measured by the level of fluorescence, are identified and selected by the present process may undergo further modifications and/or labeling/selection cycles or may be directed towards a large scale fermentation process. As such, it is desirable to include a cleavable peptide linker in the fusion peptide construct even though a cleavage step, such as acid cleavage, will not typically be included when labeling and detecting cells using the present process.
The cleavable sequence may be provided by a portion of the inclusion body tag and/or the peptide of interest (e.g., inclusion of an acid cleavable aspartic acid-proline moiety). The cleavable sequence is provided by including (in the fusion peptide) at least one cleavable peptide linker between the inclusion body tag and the peptide of interest.
Means to cleave the peptide linkers are well-known in the art and may include chemical hydrolysis, enzymatic cleavage agents, and combinations thereof. One or more chemically cleavable peptide linkers may be included in the fusion construct to facilitate recovery of the peptide of interest from the inclusion body fusion protein. Examples of chemical cleavage reagents include cyanogen bromide (cleaves methionine residues), N-chloro succinimide, iodobenzoic acid or BNPS-skatole [2-(2-nitrophenylsulfenyl)-3-methylindole] (cleaves tryptophan residues), dilute acids (cleaves at aspartyl-prolyl bonds), and hydroxylamine (cleaves at asparagine-glycine bonds at pH 9.0); see Gavit, P. and Better, M., J. Biotechnol., 79:127-136 (2000); Szoka et al., DNA, 5(1):11-20 (1986); and Walker, J. M., The Proteomics Protocols Handbook, 2005, Humana Press, Totowa, N.J.)). One or more aspartic acid-proline acid cleavable recognition sites, i.e., a cleavable peptide linker comprising one or more D-P dipeptide moieties, may be included in the fusion protein construct to facilitate separation of the inclusion body tag(s) form the peptide of interest. The fusion peptide may include multiple regions encoding peptides of interest separated by one or more cleavable peptide linkers.
One or more enzymatic cleavage sequences may be included in the fusion protein construct to facilitate recovery of the peptide of interest. Proteolytic enzymes and their respective cleavage site specificities are well known in the art. The proteolytic enzyme may be selected to specifically cleave only the peptide linker separating the inclusion body tag and the peptide of interest. Examples of enzymes useful for cleaving the peptide linker include, but are not limited to Arg-C proteinase, Asp-N endopeptidase, chymotrypsin, clostripain, enterokinase, Factor Xa, glutamyl endopeptidase, Granzyme B, Achromobacter proteinase I, pepsin, proline endopeptidase, proteinase K, Staphylococcal peptidase I, thermolysin, thrombin, trypsin, and members of the Caspase family of proteolytic enzymes (e.g. Caspases 1-10) (Walker, J. M., supra). An example of a cleavage site sequence is provided by SEQ ID NO: 170 (Caspase-3 cleavage site; Thornberry et al. J. Biol. Chem., 272:17907-17911 (1997) and Tyas et al., EMBO Reports, 1(3):266-270 (2000)).
The cleavage step, which is typically reserved for bulk processing and not used for processing the small aliquot of the cells targeted for the present labeling and detection process, occurs after the insoluble inclusion bodies and/or insoluble fusion peptides have been isolated from the cell lysate. Inclusion body processing steps are described in co-owned U.S. patent application Ser. Nos. 12/172,385 and 12/172,395; each herein incorporated by reference. The cells can be lysed using any number of means well known in the art, e.g. mechanical and/or chemical lysis. Methods to isolate the insoluble inclusion bodies/fusion peptides from the cell lysate are well known in the art, e.g., centrifugation, filtration, and combinations thereof. Once recovered from the cell lysate, the insoluble inclusion bodies and/or fusion peptides can be treated with a cleavage agent (chemical and/or enzymatic) to cleave the inclusion body tag from the peptide of interest. The fusion protein and/or inclusion body may be diluted and/or dissolved in a suitable solvent prior to treatment with the cleavage agent. The cleavage step may also be omitted if the inclusion body tag does not interfere with the activity of the peptide of interest.
After the cleavage step, the peptide of interest can be separated and/or isolated from the fusion protein and the inclusion body tags based on a differential solubility of the components. Parameters such as pH, salt concentration, and temperature may be adjusted to facilitate separation of the inclusion body tag from the peptide of interest. In one embodiment, the peptide of interest is soluble while the inclusion body tag and/or fusion protein is insoluble in the defined process matrix (typically an aqueous matrix). In one embodiment, the peptide of interest is insoluble while the inclusion body tag is soluble in the defined process matrix.
Optionally, the peptide of interest may be further purified using any number of well known purification techniques in the art such as ion exchange, gel purification techniques, and column chromatography (see U.S. Pat. No. 5,648,244), to name a few.
Fusion Peptides
The methods described herein can be used to in vivo label and optionally detect prokaryotic cells comprising any peptides/proteins having at least one tetracysteine tag capable of binding at least one biarsenical labeling reagent. The tetracysteine-tagged peptide is preferably a fusion peptide. The fusion peptides will include at least one inclusion body tag (IBT) operably linked to at least one peptide of interest (POI). Typically, the fusion peptides will also include at least one cleavable peptide linker (CL) having a cleavage site between the inclusion body tag and the peptide of interest. The inclusion body tag may include a cleavage site whereby inclusion of a separate cleavable peptide linker may not be necessary. The cleavage method may be chosen to ensure that the peptide of interest is not adversely affected by the cleavage agent(s) employed.
Any portion of the fusion peptide may contain at least one tetracysteine tag. The tetracysteine tag may be separated from the portion of the fusion protein comprising the peptide of interest by at least one at least one cleavable peptide linker. The tetracysteine tag may be operably-linked to the portion of the fusion peptide encoding the inclusion body tag. The portion of the fusion peptide comprising the IBT-TC tag may be separated from the POI by at least one cleavage peptide linker.
One of skill in the art will recognize that the fusion protein can be structured in a variety of ways. Typically, the fusion protein will include at least one IBT, at least one peptide of interest (POI), and at least one cleavable peptide linker (CL) comprising a cleavage site located between the IBT and the POI. The inclusion body tag may be organized as a leader sequence or a terminator sequence relative to the position of the peptide of interest within the fusion peptide. A plurality of IBTs, POIs, and CLs may be used when engineering the fusion peptide. The fusion peptide may include a plurality of IBTs, POIs, and cleavable peptide linkers that are the same or different.
The fusion peptide may comprise the following formula:

IBT-TC-CL-POI or POI-CL-TC-POI

wherein

IBT is at least one inclusion body tag;
TC is at least one tetracysteine tag;
CL is at least one cleavable peptide linker; and
POI is at least one peptide of interest.
The cleavable peptide linker may comprise an acid cleavable aspartic acid-proline moiety (DP).
The fusion peptide is typically insoluble in an aqueous matrix at a temperature of 10° C. to 50° C., preferably 10° C. to 40° C. The aqueous matrix typically comprises a pH range of 5 to 12, preferably 6 to 10, and most preferably 6 to 8. The temperature, pH, and/or ionic strength of the aqueous matrix may be adjusted to obtain the desired solubility characteristics of the fusion peptide/inclusion body.
In Vivo Tetracysteine Tag Labeling Using a Biarsenical Labeling Reagent
Tetracysteine tags comprising the sequence (CCXXCC; SEQ ID NO: 1) are capable of covalently binding biarsenical labeling reagents wherein the sequence CCPGCC (SEQ ID NO: 2) is preferred. Examples of suitable biarsenical labeling reagents are known in the art and include, but are not limited to FlAsh-EDT2, ReAsh-EDT2, and CHoXAsh-EDT2; available as the LUMIOTM detection system from Invitrogen Corp.; Carlsbad, Calif.; Adams et al., supra). Use of the LUMIOTM protein detection system has been reported in the art (U.S. Pat. No. 5,932,474; U.S. Pat. No. 6,054,271; U.S. Pat. No. 6,831,160; U.S. Pat. No. 6,008,378; U.S. Pat. No. 6,451,564; U.S. Pat. No. 6,686,458; U.S. Pat. No. 7,138,503; EP1032837B1, EP1684073A2, U.S. Pat. App. Pub. No. 20050176065 A1; Griffin et al., 1998, supra; Griffin et al., 2000, supra; Ho and Starnbach, supra; Adams et al., supra; Stroffekova and Proenza, supra; Rice et al., supra; and Int'l App. Pub. No. WO2007/023184A1).
However, in vivo labeling of prokaryotic host cells (Ignatova and Gierasch, et al., PNAS 101(2):523-528 (2004)) typically requires extensive dye incubation times (for example, at least 4 hours) and high concentrations (e.g., 150 μM to 1 mM) of the biarsenical labeling reagent in combination with a pretreatment step (lysozyme) to increase cell wall permeability to the labeling reagent or the presence of β-mercaptoethanol (Griffin et al., 2000, supra).
The process described herein does not require a lengthy labeling period, pretreatment with a permeabilizing agent (e.g. lysozyme, toluene), or the presence of an undesirable compound, such as β-mercaptoethanol. The present process may detect and select the labeled prokaryotic cells using a fluorescence activated cell sorter (FACS). In one embodiment, subpopulations of in vivo-labeled fluorescent cells (from the larger population of cells analyzed by FACS) are selected based on increased/higher levels of fluorescence.
Suitable labeling conditions typically include labeling approximately 106 to about 107 of properly induced bacterial cells in the fresh growth medium or PBS (phosphate buffered saline) with a suitable amount of biarsenical labeling reagent at a temperature typically ranging from about 10° C. to about 40° C., preferably about 22° C. (typical room temperature) for a period of time ranging from about 1 to less than 4 hours, and preferably about 1.5 hours to about 2 hours. The labeling period is typically conducted under low light conditions, preferably in the dark. The amount of biarsenical labeling reagent used is less than 150 μM, preferably about 5 μM to less than 150 μM, more preferably about 5 μM to about 100 μM, even more preferably 10 μM to about 50 μM, and most preferably about 10 μM to about 20 μM.
Typically, the TC-tagged peptide is under the control of an inducible expression system, such as an arabinose-inducible expression system. An effective amount of the inducer (for example, 0.2% L-arabinose for a pBAD-based expression system) is added to the media to produce the TC-tagged peptide of interest. The period of time between induction and labeling with the biarsenical labeling reagent may vary, but is typically about 5 minutes to less than 6 hours, wherein about 2.5 hours to about 4.5 hours is preferred. The biarsenical labeling reagent may be included with the inducer wherein labeling of the TC-tagged peptide occurs as the peptide is produced.
The optical density of the cells (OD600) at time of induction may vary, but typically ranges from 0.01 to about 5 prior to induction, wherein a value of 0.1 to 2 is preferred. In a preferred embodiment, the optical density (OD600) value at induction is about 0.5. The number of cells is typically normalized prior to labeling to account for difference in optimal density (OD600) between different samples. The normalized cell numbers typically range from 104 to about 108 cells, wherein about approximately 105 to about 107 of induced bacterial cells is preferred.
The labeling conditions specifically excludes, by proviso, the use of permeabilizing agents, such as lysozyme pretreatment or toluene treatment or the use of undesirable compounds, such as β-mercaptoethanol and/or other reducing agents. Pretreatments and the use of undesirable compounds increase cost and complexity to the process and may induce undesirable stress-related changes to the recombinant prokaryotic cell and/or adversely affect the labeling efficiency. The appropriate antibiotics and inducer in the labeling reaction can be maintained during the labeling process.
Detection and Isolation of In Vivo Labeled Cells Using FACS
A process is provided that combines in vivo labeling with detection and isolation of the preferred subpopulation of cells using a fluorescence activated cell sorter (FACS; Becton Dickinson). Specifically, a Becton Dickinson FACSVantageTM flow cytometry system can be used to identify, sort, and/or isolate recombinant E. coli cells comprising the fluorescently-labeled fusion peptides.
Int'l App. Pub. No. WO2007/023184A1 to Dupraz et al. describes the use of a FACS to select and identify eukaryotic cells (mammalian cell lines) expressing a chimeric selection marker comprising a tetracysteine tag (TC). However, Dupraz et al. do not describe a process to in vivo label nor identify prokaryotic cells expressing a fusion peptide comprising a tetracysteine tag (TC). No methods have been reported that describe the use of FACS to isolate prokaryotic cells expressing peptides comprising a tetracysteine tag labeled by a biarsenical labeling reagent.
Suitable FACS system parameters used to detect and sort the fluorescent cells can be determined by one of skill in the art. The excitation and emission maxima for the biarsenical dyes used in the present process are known and are described above.
FACS may be used to select a subpopulation of cells exhibiting higher fluorescence from the population of cells analyzed. The higher fluorescence is proportional to increased fusion peptide production. The process enables the identification and isolation of recombinant prokaryotic cells exhibiting increased fusion peptide production. The selected subpopulation of cells can undergo multiple rounds of selection to isolation those cells exhibiting the highest levels of fusion peptide production.
The selection parameters/criteria used to isolate a desired subpopulation using FACS may vary. Typically, the subpopulation comprises cells exhibiting the highest fluorescence with the total population assayed. In one embodiment, the top 50% of the total population of cells exhibiting fluorescence are selected (i.e. the “subpopulation”), preferably the top 25%, more preferably the top 10%, more preferably the top 5%, even more preferably the top 1%, yet even more preferably the top 0.5%, and most preferably the top 0.1%. Typically, at least 20,000-50,000 events (cells) are analyzed to set up the gates for sorting. The sorted events may vary depending upon the population of the cells available.
Evolution and Selection of Improved Production Hosts
Described herein is a process for labeling, detecting, and isolating subpopulations of live cells exhibiting improved fusion peptide production. The methods are repeatable and may be used to identify recombinant prokaryotic cells having higher and/or improved levels of recombinant fusion peptide production. The present process may include subjecting one or more of the viable recombinant prokaryotic host cells from a selected subpopulation to a procedure capable of inducing a genetic alteration (i.e., a mutagenic procedure). The mutants having beneficial mutations, such as those increasing fusion peptide production, may be isolated by repeating the present process. The process may include, for example, (1) growing the pool of mutated recombinant prokaryotic cells, (2) inducing expression of the chimeric construct whereby a fusion peptide comprising the tetracysteine tag is produced, (3) labeling the fusion peptide in vivo with a biarsenical labeling reagent, (4) detecting/isolating a subpopulation of the labeled recombinant prokaryotic host cells exhibiting increased fluorescence, and (5) amplifying the isolated cells by growing in liquid medium or on plates, optionally repeating steps (1) through (5).
Means to introduce a genetic modification to a host cell are well known in the art and may include any process that results in at least one insertion, deletion, or substitution and combinations of these to the chromosomal and/or extrachromosomal DNA within the host cell and may include mutations to one or more genes and/or portions of one or more genes (e.g. coding sequences, promoters, regulatory elements, etc.). The genetic modifications can be introduced using random or targeted mutagenesis. The genetic modification may be a disruption to an endogenous gene with the proviso that the disrupted gene is not an essential gene. For example, the genetic modification may be an insertion of a foreign gene or fragment thereof; it may be a replacement of an endogenous promoter with a heterologous promoter characterized by increased or decreased expression levels relative to the activity of the endogenous promoter; or it may be any mutation that changes relative level of transcription, translation, and/or function of a host cell gene.
Techniques for gene disruption/down-regulation are common and well known in the art of molecular biology. When the sequence of the gene to be disrupted is known, one of the most effective methods for gene down regulation is targeted gene disruption where foreign DNA is inserted into a structural gene so as to disrupt transcription. This can be accomplished by the creation of genetic cassettes comprising the DNA to be inserted (often a genetic marker) flanked by sequence having a high degree of homology to a portion of the gene to be disrupted. Introduction of the cassette into the host cell results in insertion of the foreign DNA into the structural gene via the native DNA replication mechanisms of the cell (Hamilton et al. (1989) J. Bacteriol. 171:4617-4622, Balbas et al. (1993) Gene 136:211-213, Gueldener et al. (1996) Nucleic Acids Res. 24:2519-2524, and Smith et al. (1996) Methods Mol. Cell. Biol. 5:270-277).
Whole genome sequences are available for many commercially important prokaryotic production hosts, especially bacterial strains like Escherichia coli (Baba et al., Molecular Systems Biology, article number 2006.0008, page 1-11 (2006); Blattner et al., Science, 277:1453-1462 (1997)). One of skill in the art may use targeted insertion of a nucleic acid molecule to alter the host cell. Intergenic regions between known gene clusters may be useful site when inserting an entire gene or gene cluster. Furthermore, one of skill in the art may use targeted promoter replacement to exchange promoters of varying strength to optimize the desired expression levels of certain genes (see, for example, U.S. patent application Ser. No. 10/734,936).
The λ-Red recombinase system may be used to selectively insert, disrupt and/or down-regulate expression of the desired gene. The λ-Red system works particularly well in E. coli and is very effective for targeted genetic modifications. Typically, a linear nucleic acid molecule encoding a selectable marker, such as kanamycin, is flanked by site specific recombination sequences. The linear nucleic acid molecule is designed to include sequences flanking the site specific recombination sites that have significant homology to the chromosomal gene targeted for disruption. Transformation of the construct (typically linear double stranded DNA) in the presence of the λ-Red recombinase system often results in the disruption of the targeted gene. Transformants harboring the disrupted gene are selected based on the selectable marker. A site-specific recombinase may then used (typically expressed from a curable plasmid such as pCP20; U.S. patent application Ser. No. 10/734,936) to remove the marker. Removal of the marker can be for regulatory compliance and/or to enable targeted disruption of additional genes using the same marker.
Antisense technology is another method of down-regulating genes where the sequence of the target gene is known. To accomplish this, a nucleic acid segment from the desired gene is cloned and operably-linked to a promoter such that the anti-sense strand of RNA will be transcribed. This construct is then introduced into the host cell and the antisense strand of RNA is produced. Antisense RNA inhibits gene expression by preventing the accumulation of mRNA which encodes the protein of interest. A person skilled in the art will appreciate that special considerations are associated with the use of antisense technologies in order to reduce expression of particular genes. For example, the proper level of expression of antisense genes may require the use of different chimeric genes utilizing different regulatory elements known to the skilled artisan.
Although targeted gene disruption and antisense technology offer effective means of down-regulating genes where the sequence is known, other less specific methodologies have been developed that are not sequence based. For example, cells may be exposed to UV radiation and then screened for the desired phenotype. Mutagenesis with chemical agents is also effective for generating mutants and commonly used substances include chemicals that affect non-replicating DNA such as HNO2 and NH2OH, as well as agents that affect replicating DNA such as acridine dyes, notable for causing frame-shift mutations. Specific methods for creating mutants using radiation or chemical agents are well documented in the art.
Another non-specific method of random gene insertion or random disruption is the use of transposable elements or transposons. Transposons are genetic elements that insert randomly into DNA but can be later retrieved on the basis of sequence to determine where the insertion has occurred. Both in vivo and in vitro transposition methods are known. Both methods involve the use of a transposable element in combination with a transposase enzyme. When the transposable element or transposon is contacted with a nucleic acid fragment in the presence of the transposase, the transposable element will randomly insert into the nucleic acid fragment. The technique is useful for random mutagenesis and for gene isolation, since the disrupted gene may be identified on the basis of the sequence of the transposable element. Kits for in vitro transposition are commercially available (see for example The Primer Island Transposition Kit, available from Perkin Elmer Applied Biosystems, Branchburg, N.J., based upon the yeast Ty1 element; The Genome Priming System, available from New England Biolabs, Beverly, Mass.; based upon the bacterial transposon Tn7; and the EZ::TN Transposon Insertion Systems, available from Epicentre Technologies, Madison, Wis., based upon the Tn5 bacterial transposable element). Transposon-mediated random insertion in the chromosome can be used for isolating mutants for any number of applications including enhanced production of any number of desired products including enzymes or other proteins, amino acids, or small organic molecules, such as alcohols.
Process to Detect a Prokaryotic Host Cell Having Improved Biarsenical Labeling
Depending on the cell wall/membrane structure and the intracellular environment, it may be more difficult to perform live cell labeling of certain bacteria. FACS sorting can also be used to isolate prokaryotic host cells having improved biarsenical labeling. For example, certain host mutations may increase cell permeability to facilitate the biarsenical dye to enter the cell or to improve the binding of the biarsenical dye to the target sequence. Using a library of host cells containing various mutations, it is possible to isolate the prokaryotic host mutants, which showed improved labeling efficiency. Those prokaryotic host cells may be used for future experiments that involve biarsenical labeling. The isolated mutants with increased permeability may be useful for labeling with other dyes or for other applications.
Transformation and Expression
Construction of expression cassettes and vectors suitable for use in a prokaryotic expression host is common and well known in the art. Typically, the vector or cassette contains sequences directing transcription and translation of the relevant chimeric gene, a selectable marker, and sequences allowing autonomous replication or chromosomal integration. Suitable vectors comprise a region 5′ of the gene which harbors transcriptional initiation controls and a region 3′ of the DNA fragment which controls transcriptional termination. It is most preferred when both control regions are derived from genes homologous to the transformed host cell, although it is to be understood that such control regions need not be derived from the genes native to the specific species chosen as a production host.
Transcription initiation control regions or promoters, which are useful to drive expression of the genetic constructs encoding the fusion peptides in the desired host cell, are numerous and familiar to those skilled in the art. Virtually any promoter capable of driving these constructs is suitable including, but not limited to lac, ara (pBAD), tet, trp, IPL, IPR, T7, tac, and trc (useful for expression in Escherichia coli) as well as the amy, apr, npr promoters and various phage promoters useful for expression in Bacillus. 
Termination control regions may also be derived from various genes native to the preferred hosts. Optionally, a termination site may be unnecessary; however, it is most preferred if included.
Preferred host cells for expression of the present fusion peptides are prokaryotic expression hosts found within the archaea and bacterial domains and which grow over a wide range of temperature, pH values, and solvent tolerances. For example, it is contemplated that any bacteria may be suitable hosts for expression of the present nucleic acid molecules encoding the fusion peptides. Transcription, translation, and the protein biosynthetic apparatus are universal cell process and genes are expressed regardless of the carbon feedstock used to generate the cellular biomass. Large-scale microbial growth and functional gene expression may utilize a wide range of simple or complex carbohydrates, organic acids and alcohols (i.e. methanol), saturated hydrocarbons such as methane or carbon dioxide in the case of photosynthetic or chemoautotrophic hosts. However, the functional genes may be regulated, repressed or depressed by specific growth conditions, which may include the form and amount of nitrogen, phosphorous, sulfur, oxygen, carbon or any trace micronutrient including small inorganic ions. In addition, the regulation of functional genes may be achieved by the presence or absence of specific regulatory molecules that are added to the culture and are not typically considered nutrient or energy sources. Growth rate may also be an important regulatory factor in gene expression. Examples of host strains include, but are not limited to bacterial species such as Salmonella, Bacillus, Acinetobacter, Zymomonas, Agrobacterium, Erythrobacter, Chlorobium, Chromatium, Flavobacterium, Cytophaga, Rhodobacter, Rhodococcus, Streptomyces, Brevibacterium, Corynebacteria, Mycobacterium, Deinococcus, Escherichia, Erwinia, Pantoea, Pseudomonas, Sphingomonas, Methylomonas, Methylobacter, Methylococcus, Methylosinus, Methylomicrobium, Methylocystis, Alcaligenes, Synechocystis, Synechococcus, Anabaena, Thiobacillus, Methanobacterium, Klebsiella, and Myxococcus. Preferred bacterial host strains include Escherichia, Pseudomonas, and Bacillus. The bacterial host strain may be Escherichia coli. The bacterial host strain may be derived from Escherichia coli MG 1655 (ATCC 47076 TM).
Fermentation Media
The fermentation media should contain a suitable carbon substrate. Suitable substrates include but are not limited to monosaccharides such as glucose and fructose, oligosaccharides such as lactose or sucrose, polysaccharides such as starch or cellulose or mixtures thereof and unpurified mixtures from renewable feedstocks such as cheese whey permeate, cornsteep liquor, sugar beet molasses, and barley malt. Additionally, the carbon substrate may also be one-carbon substrates such as carbon dioxide, or methanol for which metabolic conversion into key biochemical intermediates has been demonstrated. The source of carbon may include a wide variety of carbon containing substrates and is limited only by the choice of organism.
Although contemplated that all of the above mentioned carbon substrates and mixtures of these are suitable, preferred carbon substrates are glucose, fructose, and/or sucrose.
In addition to an appropriate carbon source, fermentation media must contain suitable minerals, salts, cofactors, buffers and other components, known to those skilled in the art, suitable for the growth of the cultures and promotion of the expression of the present fusion peptides.
Culture Conditions
Suitable culture conditions can be selected dependent upon the chosen production host. Typically, cells are grown at a temperature in the range of about 25° C. to about 40° C. in an appropriate medium. Suitable growth media may include common, commercially-prepared media such as Luria Bertani (LB) broth. Other defined or synthetic growth media may also be used and the appropriate medium for growth of the particular microorganism will be known by one skilled in the art of microbiology or fermentation science. The use of agents known to modulate catabolite repression directly or indirectly, e.g., cyclic adenosine 2′:3′-monophosphate, may also be incorporated into the fermentation medium.
Suitable pH ranges for the fermentation are typically between pH 5.0 to pH 9.0, where pH 6.0 to pH 8.0 is preferred.
Fermentations may be performed under aerobic or anaerobic conditions where aerobic conditions are generally preferred.
Industrial Batch and Continuous Fermentations
A classical batch fermentation is a closed system where the composition of the medium is set at the beginning of the fermentation and not subject to artificial alterations during the fermentation. Thus, at the beginning of the fermentation the medium is inoculated with the desired organism or organisms, and fermentation is permitted to occur without adding anything to the system. Typically, a “batch” fermentation is in respect to the addition of carbon source and attempts are often made at controlling factors such as pH and oxygen concentration. In batch systems the metabolite and biomass compositions of the system change constantly up to the time the fermentation is stopped. Within batch cultures, cells moderate through a static lag phase to a high growth log phase and finally to a stationary phase where growth rate is diminished or halted. If untreated, cells in the stationary phase will eventually die. Cells in log phase generally are responsible for the bulk of production of end product or intermediate.
A variation on the standard batch system is the Fed-Batch system. Fed-Batch fermentation processes are also suitable and comprise a typical batch system with the exception that the substrate is added in increments as the fermentation progresses. Fed-Batch systems are useful when catabolite repression is apt to inhibit the metabolism of the cells and where it is desirable to have limited amounts of substrate in the media. Measurement of the actual substrate concentration in Fed-Batch systems is difficult and is therefore estimated on the basis of the changes of measurable factors such as pH, dissolved oxygen and the partial pressure of waste gases such as CO2. Batch and Fed-Batch fermentations are common and well known in the art and examples may be found in Thomas D. Brock in Biotechnology: A Textbook of Industrial Microbiology, Second Edition (1989) Sinauer Associates, Inc., Sunderland, Mass. (hereinafter “Brock”), or Deshpande, Mukund V., Appl. Biochem. Biotechnol., 36:227-234 (1992).
Continuous fermentation is an open system where a defined fermentation medium is added continuously to a bioreactor and an equal amount of conditioned media is removed simultaneously for processing. Continuous fermentation generally maintains the cultures at a constant high density where cells are primarily in log phase growth.
Continuous fermentation allows for the modulation of one factor or any number of factors that affect cell growth or end product concentration. For example, one method will maintain a limiting nutrient such as the carbon source or nitrogen level at a fixed rate and allow all other parameters to moderate. In other systems a number of factors affecting growth can be altered continuously while the cell concentration, measured by media turbidity, is kept constant. Continuous systems strive to maintain steady state growth conditions and thus the cell loss due to the medium being drawn off must be balanced against the cell growth rate in the fermentation. Methods of modulating nutrients and growth factors for continuous fermentation processes as well as techniques for maximizing the rate of product formation are well known in the art of industrial microbiology and a variety of methods are detailed by Brock, supra. It is contemplated that the present process may be practiced using batch, fed-batch or continuous processes and that any known mode of fermentation would be suitable.
When an amount, concentration, or other value or parameter is given either as a range, preferred range, or a list of upper preferable values and lower preferable values, this is to be understood as specifically disclosing all ranges formed from any pair of any upper range limit or preferred value and any lower range limit or preferred value, regardless of whether ranges are separately disclosed. Where a range of numerical values is recited herein, unless otherwise stated, the range is intended to include the endpoints thereof, and all integers and fractions within the range. It is not intended that the scope of the invention be limited to the specific values recited when defining a range.
EXAMPLES
The present invention is further defined in the following Examples.
The meaning of abbreviations used is as follows: “min” means minute(s), “h” means hour(s), “μL” means microliter(s), “mL” means milliliter(s), “L” means liter(s), “nm” means nanometer(s), “mm” means millimeter(s), “μm” means micrometer(s), “μM” means micromolar, “mM” means millimolar, “M” means molar, “mmol” means millimole(s), “μmol” means micromole(s), “pmol” means picomole(s), “g” means gram(s), “μg” means microgram(s), “mg” means milligram(s), “g” means the gravitation constant, “rpm” means revolutions per minute, and “cat#” means catalog number.
General Methods
Standard recombinant DNA and molecular cloning techniques used herein are well known in the art and are described by Sambrook, J. and Russell, D., Molecular Cloning: A Laboratory Manual, Third Edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (2001); and by Silhavy, T. J., Bennan, M. L. and Enquist, L. W., Experiments with Gene Fusions, Cold Spring Harbor Laboratory Cold Press Spring Harbor, N.Y. (1984); and by Ausubel, F. M. et. al., Short Protocols in Molecular Biology, 5th Ed. Current Protocols and John Wiley and Sons, Inc., N.Y., 2002.
Materials and methods suitable for the maintenance and growth of bacterial cultures are also well known in the art. Techniques suitable for use in the following Examples may be found in Manual of Methods for General Bacteriology, Phillipp Gerhardt, R. G. E. Murray, Ralph N. Costilow, Eugene W. Nester, Willis A. Wood, Noel R. Krieg and G. Briggs Phillips, eds., American Society for Microbiology, Washington, D.C., 1994, or in Brock (supra). All reagents, restriction enzymes and materials used for the growth and maintenance of bacterial cells were obtained from BD Diagnostic Systems (Sparks, Md.), Invitrogen (Carlsbad, Calif.), Life Technologies (Rockville, Md.), QIAGEN (Valencia, Calif.) or Sigma-Aldrich Chemical Company (St. Louis, Mo.), unless otherwise specified.
DEK Medium
The composition of DEK medium is provided in Table 2.







TABLE 2







Composition of DEK medium









Final concentration















DEK medium Components





KH2PO4
9
g/L



(NH4)2HPO4
4
g/L



Citric acid *H2O
1.86
g/L



Yeast extract
5
g/L



Post sterilization



MgSO4*7H2O
1.2
g/L



Thiamine HCl
4.5
mg/L



Uracil
50
mg/L



Trace Elements Stock
10
mL/L



Trace Elements Stock Composition



EDTA
840
mg/L



CoCl2*6H2O
250
mg/L



MnCl2*4H2O
1500
mg/L



CuCl2*2H2O
150
mg/L



H3BO3
300
mg/L



Na2MoO4*2H2O
250
mg/L



Zn(CH3COO)2*2H2O
1300
mg/L



Fe(III) citrate
10000
mg/L










Design of the Expression Plasmids

Several expression systems were used to produce the fusion proteins in an E. coli host cell. One expression system was based on E. coli strain BL21-AI (Invitrogen) in combination with a T7-based expression vector wherein expression of the T7 RNA polymerase is controlled by the araBAD promoter. Another expression system was based on E. coli MG1655 (ATCC 47076TM) derived strain in combination with a pBAD-based expression vector wherein the endogenous chromosomal copy of the araBAD operon was deleted (the modified E. coli MG1655 strain comprising a disruption in the endogenous araBAD operon is referred to herein as E. coli strain KK2000).
The nucleic acid molecules encoding the various fusion peptides were designed to include at least one region encoding an inclusion body tag (IBT) linked to a peptide of interest (POI). Appropriate restriction sites were included to facilitate simple swapping of the portion encoding the inclusion body tag and/or peptide of interest. The inclusion body tag (IBT) and the peptide of interest (POI) were separated by a cleavable peptide linker (CS; for example, an acid cleavable DP moiety). Furthermore, the fusion peptide was also designed to include at least one tetracysteine tag (LUMIOTM tag; SEQ ID NO: 2) located on the C-terminus of the inclusion body tag wherein the tetracysteine tag was separated from the portion encoding the peptide of interest by the cleavable peptide linker.
Expression Plasmid pTG28 (also referred to herein as pTG028; SEQ ID NO: 19)) is a ColE1 plasmid that has a pBAD promoter expressing the fusion peptide containing the inclusion body tag KSI(C4) and HC77643. It also has the araC gene encoding the regulator and the bla gene conferring ampicillin resistance.
Expression Plasmid pTG34 (also referred to herein as pTG034; SEQ ID NO: 20) is a ColE1 plasmid that has a T7 promoter expressing the fusion peptide containing the inclusion body tag IBT139 and HC77643. It also has the bla gene conferring ampicillin resistance.
Expression Plasmid pLR173 (SEQ ID NO: 21) is a ColE1 plasmid that has a pBAD promoter expressing the fusion peptide containing the inclusion body tag IBT139 and HC77643. It also has araC encoding the regulator, bla conferring ampicillin resistance and the aadA-1 gene conferring spectinomycin resistance.
Plasmid pLR186 (SEQ ID NO: 18) contains a ColE1 type origin of replication, the bla gene to confer ampicillin resistance and the aadA-1 gene to confer spectinomycin (Spec) resistance. The fusion peptide containing inclusion body tag IBT139 fused with peptide HC124 (see U.S. patent application Ser. No. 11/782,836) was expressed by the pBAD promoter. The plasmid also encodes the gene for the araC regulator.
Plasmid pLR199 (FIG. 1D; SEQ ID NO: 22) was created from pLR186 by inserting an annealed oligonucleotide fragment encoding a tetracysteine tag CCPGCC (SEQ ID NO: 2) into the BamHI site between the inclusion body tag IBT139 (SEQ ID NO: 12) and HC776124 (SEQ ID NO: 10). The resulting fusion peptide was IBT139.CCPGCC-HC776124. Inclusion body tag IBT139.CCPGCC is provided as SEQ ID NOs: 13-14.
FACS System Operating Conditions:
A Fluorescence Activated Cell Sorter (FACSVantageTM SE-DiVa; Becton-Dickinson (BD Biosciences, Franklin Lakes, N.J.)) was configured with a single 488 nm argon ion laser (200 mW). The laser is used to induce light scattering by either the excitation of cellular fluorescent tags or the granularity within the cell. The SSC (Side Scatter Collector) light detection from the cell is collected through a microscope objective, transmitted via fiber light guide to an array of photo-multiplier tubes (PMT's). The FSC (Forward Scatter Collector) was constructed of a photo-diode. The SSC octagon configuration was composed of 5 PMT's in an octagon configuration. The LUMIOTM collection at 530 nm used a fluorescein isothiocyanate (FITC) filter (530 nm center, +/−15 nm bands) with a SSC filter of 488 nm bandpass (488 nm center, +/−10 nm bands). The system fluid used on the FACSVantageTM SE-DiVa was FACSFlowTM Sheath (Becton Dickinson) at an operating pressure of 28 psi ( ̃193 kPa) using a 70 μm diameter orifice tip.
The standard daily alignment of the instrument was performed using ALIGNFLOWTM (Molecular Probes, Inc., Eugene, Oreg.) 2.5 μm diameter fluorescent beads at an excitation/emission of 488 nm. The ALIGNFLOWTM beads were used as the daily alignment standard and the following instrument adjustments were made on the FACS to obtain the maximum PMT signal and minimum CV (coefficient of variation) for all channels on the instrument. The ALIGNFLOWTM beads were used to enable the daily adjustment of the FACS nozzle (X, Y, Z, α, and θ); in addition to the focus lens, channel height and channel height focus in all detector channels. The alignment of the FACS system can vary, but with the use of the ALIGNFLOWTM beads, good alignment reproducibility was obtained. The ALIGNFLOWTM beads were either incorporated as a separate sample or directly into the sample to monitor the alignment and any potential instrument drift. The daily FACS alignment procedure, created in the DiVa Software (Becton Dickinson, v1.4), was performed and verified to within normal operating conditions.
The LUMIOTM stained cell samples were previously prepared in PBS (Phosphate Buffered Saline) which is similar to the sheath fluid; therefore, no additional manipulation was needed for FACS analysis. Approximately 200 μL of a sample containing LUMIOTM stained cells was placed into a Falcon 12×75 mm, sterile polystyrene culture tube (Becton Dickinson) and into the instrument. The sample differential pressure was adjusted to obtain a stable 1000 events/second; at which, between 20,000 and 50,000 sample events were recorded. The variation, in sample recorded events, was due to the variation in cell concentration and limited sample volume. If the observed events/s were observed to be low, then the recorded events were then decreased. The samples scanned on the FACS for LUMIOTM analysis included, but were not limited to, an ALIGNFLOWTM bead sample, unstained LUMIOTM (negative control) and a series of LUMIOTM stained samples (experimental). The data obtained for the FACS samples included several different plot windows; which included dot plots for FSC-A vs. SSC-A, FSC-A vs. FITC-A, SSC-A vs. FITC-A and histograms for SSC-A, FSC-A, and FITC-A (width×height) for the particular channel (“A” is the computed area; “FS” is forward scatter; and “SS” is side scatter). During the recording of each sample, a gate was set on the FITC-A histogram between the 103 and 104 (log scale) to monitor and observe the sample LUMIOTM labeling efficiency. The recorded events within the gate on the FITC-A log scale provided a good indication of the sample LUMIOTM labeling efficiency. The recorded LUMIOTM sample data was saved and then within the DiVA software they were exported as FCS3 data files for further analysis.
Example 1
Specific Labeling of Tetracysteine Tagged Fusion Peptides
This example describes cloning of the tetracysteine tag (CCPGCC; SEQ ID NO: 2) into three peptide expression plasmids in E. coli, which allowed specific labeling of the fusion peptides.
Specific labeling of proteins or peptides could be achieved by biarsenical ligands binding to tetracysteine tag (CCX1X2CC, SEQ ID NO: 1; wherein X1 and X2 are any amino acid other than cysteine, preferably X1X2 is PG; respectively (SEQ ID NO: 2)), which is genetically engineered into the target protein or peptide. The fluorescein derivative with two As(III) substituents, FlAsH-EDT2 (LUMIOTM Green), only fluoresces after the arsenics bind to the cysteine thiols in the target protein or peptide. In order to reach high production yield and reduce separation costs, peptides of interest were produced as inclusion bodies in our E. coli production hosts. Inclusion body promoting sequences were usually fused to the N-terminus of the peptide of interest to drive peptide production into inclusion bodies. An acid cleavage site (DP) was introduced after the inclusion body promoting sequence to separate the inclusion body promoting sequence away from the peptide of interest after production. At least one tetracysteine tag (LUMIOTM tag; SEQ ID NO: 2) was cloned onto the C-terminus of the inclusion body tag wherein the tetracysteine tag was separated from the portion encoding the peptide of interest by the cleavable peptide linker. The LUMIOTM reagents used for fluorescent labeling were obtained from Invitrogen (Carlsbad, Calif.).
The oligonucleotides encoding the tetracysteine tag CCPGCC (SEQ ID NO: 2) were synthesized by Sigma Genosys (The Woodlands, Tex.). The top strand oligo 5′-GA TCT TGC TGT CCG GGC TGT TGC G-3′ (SEQ ID NO: 3) and the bottom strand oligo 5′-GA TCC GCA ACA GCC CGG ACA GCA A-3′ (SEQ ID NO: 4) were annealed with a Bglll overhang at the 5′ end and a BamHI overhang at the 3′ end. The annealed double stranded fragment was cloned into the BamHI site of a peptide expression plasmid pTG28, which contained a nucleic acid molecule encoding peptide of interest HC77643 (SEQ ID NO: 8) fused to the inclusion body promoting sequence KSI(C4) (SEQ ID NO: 16) expressed by the PBAD promoter. The resulting clone pDCQ500 (FIG. 1A; SEQ ID NO: 23) contained the tetracysteine tag inserted after the inclusion body promoting sequence and before the acid cleavage site (FIG. 1E). The tetracysteine tag was also cloned similarly into two other peptide expression plasmids pTG34 (SEQ ID NO: 20) and pLR173 (SEQ ID NO: 21). Plasmid pLR173 contains a chimeric gene encoding the peptide of interest (HC77643; SEQ ID NO: 8) fused to a different inclusion body promoting sequence IBT139 (SEQ ID NO: 12) expressed by the PBAD promoter. Plasmid pTG34 contains a chimeric gene encoding the peptide of interest HC77643 fused to the IBT139 expressed by the T7 promoter. The tetracysteine tag was inserted between the IBT139 and HC77643 by cloning into the BamHI site of the parental plasmids pTG34 and pLR173, resulting in plasmids pDCQ502 (FIG. 1B; SEQ ID NO: 24) and plasmid pDCQ506 (FIG. 1C; SEQ ID NO: 25), respectively. Plasmids pDCQ500 and pDCQ506 were transformed into an E. coli K-derived production host KK2000 (MG1555 ΔaraBAD). Plasmid pDCQ502 was transformed into an E. coli B-derived production host BL21-AI (Invitrogen).
The cultures were grown from fresh overnight cultures to OD600 0.3-0.5, and were then induced with 0.2% L-arabinose (Sigma) for 3 to 4 hours at 37° C. Cells from 100 μL cultures were pelleted and resuspended in 50 μL B-Per lysis buffer (Pierce) or the lysis buffer containing 50 mM K phosphate buffer pH 7.8, 400 mM NaCl, 100 mM KCl, 10% glycerol, 0.5% Triton X-100, 10 mM imidazole, 1 mg/mL lysozyme (Sigma) and 13.6 units of DNase I (Qiagen, Valencia, Calif.). The lysates were spun at 14,000×g for 5 min to separate the supernatant and the inclusion body fraction. The inclusion body pellets were washed and resuspended in 8 M urea. The whole lysate or the separated supernatant and inclusion body fractions were stained with LUMIOTM Green detection reagent using the LUMIOTM Green detection kit (Invitrogen) following manufacturers' instructions. The samples were run on a NuPAGE® 4-12% Bis-Tris gel with the BenchMarkTM fluorescent protein standard (Invitrogen). The gel was visualized under UV light. After taking a picture, the gel was rinsed, stained with SimplyBlueTM (Invitrogen) and destained with deionized water. The fusion peptides were specifically labeled in the lysates from strains containing the tetracysteine tag, whether the peptides were fused with the KSI(C4) or IBT139 inclusion body promoting sequences, and expressed by PBAD promoter in KK2000 strain or T7 promoter in BL21-AI strain. No protein was stained in the lysates from strains without the tetracysteine tag. Incorporation of one copy of the tetracysteine tag into these peptides neither affected the peptide expression nor the peptide partition in the production hosts.
Example 2
In Vivo Fluorescent Labeling of E. coli Cells Producing the Tagged Peptides Visualized by Fluorescence Microscope
To explore if the live E. coli cells producing the tetracysteine-tagged peptides could be labeled in vivo without cell lysis or membrane permeabilization, we performed the labeling of the live E. coli cells using the LUMIOTM in-cell labeling kit (catalog no. 12589-057) or the TC-FlAsHTM TC-ReAsHTM II in-cell tetracysteine tag detection kit (catalog no. T34561) from Invitrogen, which are designed for labeling mammalian cells in vivo. The E. coli cells were grown and induced as described above. Normalized amounts of 100-200 μL of OD600 0.01 cells were pelleted and used in labeling reactions containing 1×PBS, 0.2% L-arabinose, appropriate antibiotics, 1 μM TCEP (Tris[2-carboxyethyl]phosphine) and 10-20 μM LUMIOTM reagent. In the only report of LUMIO In-Cell labeling in E. coli, Ignatova and Gierasch (PNAS 101 (2):523-528, 2004) used much higher concentrations of the labeling reagent (up to 1 mM). The reactions were incubated at room temperature in the dark for 90-120 min. The cells were washed twice with PBS containing 20 μM Disperse Blue 3 (1-((2-Hydroxyethyl)amino)-4-(methylamino) anthraquinone; CAS# 2475-46-9) or 250 μM BAL wash buffer provided in the kits, and visualized under a fluorescent light microscope with the appropriate filter for fluorescein isothiocyanate (FITC). Fluorescent staining was observed specific for the inclusion bodies usually found at the distal ends of the cells. Minimal background was observed for uninduced cells not producing any tagged fusion peptide.
Viability of cells was also checked with the LIVE/DEAD® BacLight® bacterial viability kit (by Molecular Probes, available from Invitrogen), which utilizes the fluorescent nucleic acid stains SYTO® 9 (Molecular Probes) and propidium iodide. A fraction of induced cells was stained in suspension according to the manufacturer's instructions and examined by fluorescent light microscope using the broad band filter. Vast majority of cells fluoresced green, which indicated that the induced peptide-producing cells were still viable. The LUMIOTM in cell labeling was able to stain the inclusion bodies in the live E. coli cells without membrane permeabilization.
Example 3
FACS Analysis of LUMIOTM-Labeled E. coli B Cells
E. coli BL21-AI cells containing the pDCQ502 and the parent pTG34 plasmids were grown to OD600 about 0.5 and either continue to grow as un-induced or induced with 0.2% L-arabinose for 2.5 hours. Cells were then normalized to OD600 of 0.01 and were labeled by LUMIOTM in vivo as described in Example 2. Approximately 105-106 labeled cells were analyzed with a FACS VantageTM SE with Digital Vantage manufactured by Becton Dickinson (BD Biosciences, Franklin Lakes, N.J.). The FACS instrument was configured and aligned as described in the General Methods. Twenty-thousand events were recorded for each sample. Data summarized in Table 3 showed that E. coli BL21-AI cells containing the pTG34 plasmid (i.e. the plasmid does not contain the nucleic acid sequence encoding the tetracysteine tag) were not labeled by LUMIOTM Green. Induced BL21-AI cells containing the pDCQ502 plasmid encoding a fusion peptide comprising the tetracysteine tag (CCPGCC; SEQ ID NO: 2) were labeled by LUMIOTM Green. Uninduced cells not making the peptide were not labeled. This suggested that LUMIO Green specifically labeled the tagged peptide in vivo and the fluorescent intensity generated by LUMIOTM labeling is sensitive for FACS analysis.







TABLE 3







Percentage of labeled cells by FACS analysis











% labeled cells



Strain
(FITC 102-104)














BL21-AI (pTG34) uninduced
6.8



BL21-AI (pTG34) induced
10.5



BL21-AI (pDCQ502) uninduced
4.5



BL21-AI (pDCQ502) induced
91.2










Example 4
FACS Analysis of LUMIOTM Labeled E. coli K Cells
It was reported (Stroffekova, K., et al, Pflugers Arch-Eur J Physiol, 442:859-866 (2001)) that the LUMIOTM labeling reagent binds not only to CCXXCC (SEQ ID NO: 1) motifs, but also non-specifically to endogenous cysteine-rich proteins. E. coli MG1655 SlyD is a cysteine-rich protein that might contribute to the background of LUMIOTM labeling (Keppetipola, S., et al, Focus 25.3:7-11 (2003) and U.S. Pat. App. Pub. US20050136449). A SlyD deletion strain was constructed in KK2000 by P1 transduction using general protocols as outlined in Experiments in Molecular Genetics, Miller, J. H., 1972, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. The SlyD deletion strain JW3311 in the Keio collection (Baba, T., et al, 2006, Molecular Systems Biology, 1-11; article #2006.0008) was used as the donor. The kanamycin resistant P1 phage was isolated and was used to transduce the KK2000 recipient. The kanamycin resistant marker flanked by FLP sites was then removed by transient expression of the Flp recombinase on a temperature-sensitive plasmid pCP20 (ATCC PTA-4455; U.S. patent application Ser. No. 10/734,936; and Cherepanov and Wackernagel (Gene, 158:9-14 (1995)). Loss of kanamycin resistance marker and deletion of slyD in KK2000 was confirmed by PCR. The resulting strain was designated as QC1100.
Plasmid pLR199 or pLR186 was transformed into QC1100. Plasmid pLR199 (FIG. 1D; SEQ ID NO: 22) comprises a chimeric gene encoding the fusion peptide of IBT139.CCPGCC-HC776124 expressed by pBAD promoter. HC776124 (SEQ ID NO: 10) is a dimer of HC77643 linked together by Pro-Gly linker. Plasmid pLR186 was the parent of pLR199 without the tetracysteine tag. E. coli QC1100(pLR199) or QC1100(pLR186) cells were grown to OD600 about 0.5 and induced with 0.2%, 0.02%, 0.002%, 0.0002% or 0% L-arabinose for 2.5 hours. Cells were then normalized to OD600 of 0.01. Approximately 105-106 cells were labeled by LUMIOTM and analyzed by FACS as described in Example 2. FACS analysis showed that uninduced cells or the vast majority of cells induced with low concentrations of arabinose (0.0002% or 0.002%) were not labeled. Cells induced with high concentrations of arabinose (0.02% or 0.2%) were labeled. Cells with and without the tetracysteine tag were both labeled, which cells with the tag showed higher fluorescent intensity. Table 3 summarized percentage of fluorescent labeled cells within the 103-104 gate and within the 102-103 gate. The fluorescent labeling of the peptide without the tetracysteine tag might be due to nonspecific labeling of the inclusion body structure of the fusion peptide.







TABLE 4







Percentage of labeled cells by FACS analysis










% QC1100(pLR199)
% QC1100(pLR186)



cells
cells












FITC

FITC



% L-arabinose
102-103
FITC 103-104
102-103
FITC 103-104














0
1.8
0.04
1.2
0.1


0.0002
4.4
0.2
0.8
0.06


0.002
37.2
1.4
7.0
0.1


0.02
37.8
51.1
7.4
1.9


0.2
21.3
65.6
76.7
6.3









The high fluorescent 1% and low fluorescent 1% cells were collected from QC1100(pLR199) induced with 0.2% L-arabinose. They were plated onto LB plates containing 100 μg/mL ampicillin. Similar numbers of colonies were recovered from the high fluorescent and low fluorescent fractions. The ability of obtaining viable cells after LUMIOTM labeling and sorting indicates that the labeling process was suitable for FACS sorting.

Example 5
FACS Sorting of High Fluorescent Cells Based on LUMIOTM Labeling
This example demonstrates that using FACS based on fluorescence generated from LUMIOTM labeling, it was possible to sort out high fluorescent cells from low fluorescent cells. This could be exploited to isolate cells producing higher titers of peptides.
E. coli QC1100(pLR199) cells and QC1100(pLR186) cells were grown to OD600 about 0.5 and induced with 0.2% L-arabinose for 2.5 hours. The induced cells from the two strains were mixed in 1:1 ratio. Approximately 105-106 of mixed cells as well as cells of the two individual strains were labeled by LUMIOTM and analyzed by FACS as described in Example 4. Cells of QC1100(pLR199) showed a peak of higher fluorescence with mean of fluorescent intensity as 1387. Cells of QC1100(pLR186) showed a peak of lower fluorescence with mean of fluorescent intensity as 778. Cells from the mixture showed two overlapping peaks indicating of the mixed population.
The 1% high fluorescent cells from the mixed population were collected on LB plates with ampicillin (100 μg/mL) and grown at 37° C. overnight. Approximately 1270 colonies were produced on the plates. Twenty-two colonies were randomly picked for in-gel labeling as described in Example 1 to identify the isolated strains. The LUMIOTM tagged fusion protein in the extract of QC1100(pLR199) cells could be fluorescently labeled in the gel. The peptide without the LUMIOTM tag in the extract of QC1100(pLR186) cells could not be fluorescently labeled in the gel. Results showed that among the 22 colonies randomly picked, all were QC1100(pLR199). This demonstrated that the highly fluorescent QC1100(pLR199) cells can be sorted out from the mixture by a single round of FACS sorting based on LUMIOTM in-cell labeling.
Example 6
Increased LUMIOTM In-Cell Labeling Reflected Increased Peptide Production
QC1100 cells expressing the fusion peptide IBT139-CCPGCC-HC776124 with the tetracysteine tag were grown from fresh overnight cultures for 1.5 hours to OD600  ̃0.5 and induced with 0.2% L-arabinose for different lengths of time (0, 0.5, 1, 1.5, 2, and 2.5 hours). Aliquots of the induced cultures were diluted and approximately 3×106 cells were used for in-cell labeling as described above. The labeled cells were washed and subjected for FACS analysis. Additional aliquots of the induced cultures of OD600  ̃1 were normalized and pelleted for in-gel labeling as described in Example 1. The labeled peptides were separated on a NUPAGE® 4-12% Bis-Tris gel and visualized under UV light. FIGS. 2A-2F show the FACS analysis results at different induction times. FIGS. 2G and 2H show the corresponding peptide gel analysis results. As the induction time increases, cells produced more peptide as shown by the increased fluorescence intensity of the peptide product on the gel. FACS analysis also showed the increased fluorescent intensity at a single cell level, which reflected increased peptide production. The non-linear increase of fluorescent intensity by FACS was most likely due to the all-or-non nature of arabinose induction at the single cell level (Siegele, D. A. & Hu, J. C. (1997) Proc. Natl. Acad. Sci. USA 94:8168-8172).
Example 7
FACS Analysis of LUMIOTM Labeled E. coli Cells Grown in DEK Medium
This example describes LUMIOTM in-cell labeling of E. coli cells expressing the fusion peptide containing the tetracysteine tag grown in a minimal based DEK medium (Table 2).
QC1100 cells expressing the fusion peptide IBT139-CCPGCC-HC776124 with the tetracysteine tag were grown in LB with appropriate antibiotics overnight. The cells were subcultured 1:100 from fresh overnight cultures into DEK medium (see General Methods for composition). Growth in DEK medium was monitored and aliquots of cells were taken out for induction with 0.2% L-arabinose at different OD600. After postinduction growth for 3 hours, aliquots of the induced cultures were diluted and approximately 3×106 cells were used for in-cell labeling in labeling reactions containing 1×DEK medium, 0.2% L-arabinose, appropriate antibiotics, 1 μM TCEP (Tris[2-carboxyethyl]phosphine) and 20 μM LUMIOTM reagent. The labeled cells were washed and subjected to FACS analysis. Results showed that cells were not highly labeled if they were induced at low OD600 values (<1.0). Cells were better labeled if they were induced at OD600>1.0. Even with the optimal labeling as shown in FIG. 3A, DEK grown cells showed lower fluorescence intensity (mean  ̃500) than the LB grown cells (mean of fluorescent intensity  ̃1500; FIG. 3B). Normalized amount of cells from the induced cultures were also used for in-gel labeling. It appeared that DEK grown cells produced less peptide than the LB grown cells. This was consistent with the FACS data showing lower fluorescent intensity of the labeled DEK grown cells.
LUMIOTM in-cell labeling of E. coli cells expressing the fusion peptide containing the tetracysteine tag was also achieved in other rich media such as 2xYT or other minimal media such as M9.

## Claims

1. A process for in vivo labeling and detecting a polypeptide or protein in a prokaryotic cell comprising:
a) providing an unpermeabilized prokaryotic host cell comprising a chimeric genetic construct encoding a polypeptide or protein comprising at least one tetracysteine tag;
b) growing the unpermeabilized prokaryotic host cell of (a) under suitable conditions to express the chimeric genetic construct and to produce the polypeptide or protein comprising said at least one tetracysteine tag;
c) contacting in vivo the prokaryotic host cell of (b) with an effective amount of at least one biarsenical labeling reagent that binds to the at least one tetracysteine tag forming at least one fluorescent complex, wherein said contacting is performed in the absence of a reducing agent; and
d) detecting the prokaryotic cell comprising the at least one fluorescent complex by utilizing a fluorescence activated cell sorter (FACS) to select a subpopulation of cells which display the highest fluorescence.

2. The process of claim 1, whereby the chimeric genetic construct encodes a fusion peptide comprising:
i) at least one first portion comprising an inclusion body tag (IBT);
ii) at least one second portion comprising a peptide of interest (POI); and
iii) at least one tetracysteine tag (TC).

3. The process of claim 1, wherein the prokaryotic host cell is an enteric bacteria.

4. The process of claim 1, wherein the effective amount of the at least one biarsenical labeling reagent ranges from about 5 μM to less than 150 μM.

5. The process of claim 1, wherein the tetracysteine tag comprises the amino acid sequence of SEQ ID NO: 2.

6. The process of claim 1, wherein the at least one biarsenical labeling reagent is selected from the group consisting of fluorescein arsenical hairpin binding reagent and resorufin arsenical hairpin binding reagent.

7. The process of claim 2, wherein the genetic construct further comprises a nucleic acid molecule encoding a cleavable peptide linker located between the tetracysteine tag and the peptide of interest.

8. The process of claim 7, wherein the at least one first portion comprising the inclusion body tag comprises the at least one tetracysteine tag.

9. The process of claim 1, wherein steps (a) through (d) are optionally repeated.

10. The process of claim 1, further comprising the step of:
(e) subjecting the selected subpopulation to at least one mutagenic procedure capable of introducing at least one genetic alteration to the selected cell which increases the production of said tetracysteine tagged polypeptide or protein.

11. The process of claim 10, wherein steps (a) through (e) are optionally repeated.

12. The process of claim 2 wherein said fusion peptide comprises the structure:


IBT-TC-CL-POI or POI-CL-TC-IBT

wherein
a) CL is a cleavable peptide linker;
b) the tetracysteine tag comprises amino acid sequence SEQ ID NO: 2;
c) the biarsenical labeling reagent is selected from the group consisting of fluorescein arsenical hairpin binding reagent and resorufin arsenical hairpin binding reagent; and
d) the prokaryotic cell is Escherichia coli.

13. A process for making an improved fusion protein comprising:
a) providing an unpermeabilized prokaryotic host cell comprising a chimeric genetic construct encoding a polypeptide or protein comprising at least one tetracysteine tag;
b) subjecting the unpermeabilized prokaryotic cell of (a) to a mutagenic procedure to produce a pool of mutagenized prokaryotic cells which display increased tetracysteine tagged polypeptide or protein production;
c) inducing expression of the chimeric genetic construct to produce a fusion peptide comprising the tetracysteine tag;
d) labeling the fusion peptide in vivo with a biarsenical labeling reagent in the absence of a reducing agent;
e) isolating a subpopulation of the labeled, unpermeabilized prokaryotic cells exhibiting increased fluorescence; and
f) amplifying the isolated prokaryotic cells by growing in liquid medium or on plates; and
g) optionally repeating steps (b)-(f).