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WO2005095988A2 - Selection de polypeptides d'ancrage a une membrane interne bacterienne - Google Patents

Selection de polypeptides d'ancrage a une membrane interne bacterienne Download PDF

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Publication number
WO2005095988A2
WO2005095988A2 PCT/US2005/009009 US2005009009W WO2005095988A2 WO 2005095988 A2 WO2005095988 A2 WO 2005095988A2 US 2005009009 W US2005009009 W US 2005009009W WO 2005095988 A2 WO2005095988 A2 WO 2005095988A2
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WIPO (PCT)
Prior art keywords
inner membrane
polypeptide
bacterium
cell
heterologous polypeptide
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WO2005095988A3 (fr
Inventor
George Georgiou
Barrett R. Harvey
Ki Jun Jeong
Brent L. Iverson
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University of Texas System
University of Texas at Austin
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University of Texas System
University of Texas at Austin
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Publication of WO2005095988A3 publication Critical patent/WO2005095988A3/fr
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/10—Processes for the isolation, preparation or purification of DNA or RNA
    • C12N15/1034—Isolating an individual clone by screening libraries
    • C12N15/1086—Preparation or screening of expression libraries, e.g. reporter assays
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/10—Processes for the isolation, preparation or purification of DNA or RNA
    • C12N15/1034—Isolating an individual clone by screening libraries
    • C12N15/1037—Screening libraries presented on the surface of microorganisms, e.g. phage display, E. coli display
    • C—CHEMISTRY; METALLURGY
    • C40—COMBINATORIAL TECHNOLOGY
    • C40B—COMBINATORIAL CHEMISTRY; LIBRARIES, e.g. CHEMICAL LIBRARIES
    • C40B40/00—Libraries per se, e.g. arrays, mixtures
    • C40B40/02—Libraries contained in or displayed by microorganisms, e.g. bacteria or animal cells; Libraries contained in or displayed by vectors, e.g. plasmids; Libraries containing only microorganisms or vectors
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/01—Fusion polypeptide containing a localisation/targetting motif
    • C07K2319/034—Fusion polypeptide containing a localisation/targetting motif containing a motif for targeting to the periplasmic space of Gram negative bacteria as a soluble protein, i.e. signal sequence should be cleaved

Definitions

  • Ligand-binding polypeptides including proteins and enzymes with a desired substrate specificity can be isolated from large libraries of mutants, provided that a suitable screening method is available. Small protein libraries composed of 10 3 -10 5 distinct mutants can be screened by first growing each clone separately and then using a conventional assay for detecting clones that exhibit specific binding. For example, individual clones expressing different protein mutants can be grown in microtiter well plates or separate colonies on semisolid media such as agar plates.
  • the underlying premise of display technologies is that proteins engineered to be anchored on the external surface of biological particles (i.e., cells or viruses) are directly accessible for binding to ligands without the need for lysing the cells.
  • Viruses or cells displaying proteins with affinity for a ligand can be isolated in a variety of ways including sequential adsorption/desorption form immobilized ligand, by magnetic separations or by flow cytometry (Ladner et al. 1993, U.S. Patent 5,223,409, Ladner et al. 1998, US patent 5,837,500, Georgiou et al. 1997, Shusta et al. 1999).
  • the most widely used display technology for protein library screening applications is phage display.
  • Phage display is a well-established and powerful technique for the discovery of proteins that bind to specific ligands and for the engineering of binding affinity and specificity (Rodi and Makowski, 1999).
  • phage display a gene of interest is fused in-frame to phage genes encoding surface-exposed proteins, most commonly pill. The gene fusions are translated into chimeric proteins in which the two domains fold independently.
  • Phage displaying a protein with binding affinity for a ligand can be readily enriched by selective adsorption onto immobilized ligand, a process known as "panning". The bound phage is desorbed from the surface, usually by acid elution, and amplified through infection of E. coli cells.
  • phage display imposes minimal selection for proper expression in bacteria by virtue of the low expression levels of antibody fragment gene III fusion necessary to allow phage assembly and yet sustain cell growth (Krebber et al, 1996, 1997). As a result, the clones isolated after several rounds of panning are frequently difficult to produce on a preparative scale in E. coli.
  • phage displayed proteins may bind a ligand, in some cases their un-fused soluble counterparts may not (Griep et ah, 1999).
  • the isolation of ligand-binding proteins and more specifically antibodies having high binding affinities can be complicated by avidity effects by virtue of the need for gene III protein to be present at around 5 copies per virion to complete phage assembly.
  • the invention provides a method of obtaining a bacterium comprising a nucleic acid sequence encoding an inner membrane anchor polypeptide capable of anchoring a heterologous polypeptide to the outer side of the inner membrane of a Gram negative bacterium comprising the steps of: (a) providing a Gram negative bacterium comprising an inner membrane, an outer membrane and a periplasm; the bacterium comprising a nucleic acid sequence encoding a fusion between a heterologous polypeptide and a candidate inner membrane anchor sequence; (b) removing the outer membrane; and (c) selecting the bacterium based on the presence of the heterologous polypeptide anchored to the outer side of the inner membrane to identify an inner membrane anchor polypeptide capable of anchoring a heterologous polypeptide to the outer side of the inner membrane of the bacterium.
  • the method may be further defined as a method of obtaining a nucleic acid sequence encoding an inner membrane anchor sequence capable of anchoring a heterologous polypeptide to the outer side of the inner membrane, the method further comprising the step of: (d) cloning a nucleic acid sequence encoding the inner membrane anchor polypeptide.
  • Selecting the bacterium may comprise detecting the heterologous polypeptide with a binding polypeptide having specific affinity for the heterologous polypeptide. This may further comprise use of at least a second binding polypeptide having affinity for the heterologous polypeptide and/or the binding polypeptide having specific affinity for the heterologous polypeptide.
  • the second binding polypeptide may be an antibody or fragment thereof, which may be fluorescently or otherwise labeled. Selecting the bacterium may comprise use of at least a third binding polypeptide having specific affinity for the heterologous polypeptide and/or the second binding polypeptide to label the bacterium.
  • the heterologous polypeptide may, comprise a detectable label. Examples of detectable labels include an antigen and GFP.
  • the heterologous polypeptide may also comprise an antibody or fragment thereof. Selecting the bacterium may comprise detecting the antibody or fragment thereof with a labeled ligand having specific affinity for the antibody or fragment thereof.
  • the Gram negative bacterium is an E. coli bacterium.
  • step (a) is further defined as comprising providing a population of Gram negative bacteria. - ⁇
  • the population of bacteria ay be further defined as collectively expressing a plurality of candidate .inner membrane anchor sequences.
  • the bacterium may be viable or non-viable.
  • Cloning may comprise amplification of the nucleic acid sequence. Selecting may be carried out by flow-cytometry or magnetic separation.
  • the nucleic acid encoding a candidate inner membrane anchor polypeptide may be flanked by known PCR primer sites.
  • the candidate inner membrane anchor polypeptide may be anchored to the outer side of the inner membrane with a transmembrane protein or fragment thereof.
  • the transmembrane protein or fragment thereof may comprise a sequence selected from the group consisting of: the first two amino acids encoded by the E. coli NlpA gene, the first six amino acids encoded by the E.
  • the candidate inner membrane anchor polypeptide may be anchored via an N- or C-terminus of the polypeptide.
  • the candidate inner membrane anchor polypeptide sequence may be anchored to the outer side of the inner membrane with an inner membrane lipoprotein or fragment thereof selected from the group consisting of: AraH, MglC, MalF, MalG, Mai C, MalD, RbsC, RbsC, ArtM, ArtQ, GlnP, ProW, HisM, HisQ, LivH, LivM, LivA, Liv E,Dpp B, DppC, Op ⁇ B,AmiC, AmiD, BtuC, FhuB, FecC, FecD,FecR, FepD, NikB, NikC, CysT, CysW, UgpA, UgpE, PstA, PstC, Pot
  • the invention provides a method of obtaining a bacterium 5 comprising a nucleic acid sequence encoding an inner membrane anchor polypeptide capable of anchoring a heterologous polypeptide to the outer side of the inner membrane of a Gram negative bacterium comprising the steps of: (a) providing a population of Gram negative bacteria the members of which comprise an inner membrane, an outer membrane and a periplasm; wherein the bacteria collectively comprise nucleic acid sequences encoding fusion
  • step (c) may be further defined as selecting a subpopulation of bacteria comprising the heterologous polypeptide anchored to the outer side
  • Step (c) may also comprise fluorescently labeling the heterologousi . : ', ' r polypeptide followed by fluorescence activated cell sorting;(F ACS). ,' . : . ' > i. . ⁇ ⁇
  • FIG. 1A-C Selective identification of Antigen targets with APEx.
  • FIG. 1 A digoxigenin-Bodipy FL
  • FIG. IB methamphetamine-FL
  • FIG. 1C ScFvs expressed that bind peptides
  • FIG. 2A-B Detection of ScFvs on the Surface of Spheroplasts.
  • FIG. 3A-B Detection of ScFvs for Larger Target Antigen conjugated fluorophores.
  • FIG. 5 Analysis of clone designated mutant 9 with higher mean FL signal than the parent anti-methamphetamine scFv.
  • the scFvs expressed via anchored periplasmic expression are as indicated.
  • FIG. 6 A schematic diagram showing the principle of Anchored Periplasmic Expression (APEx) for the flow cytometry based isolation of high affinity antibody fragments.
  • FIG. 7 Examples of targets visualized by APEx.
  • FIG. 7 A Fluorescence distribution of ABLECTM cells expressing PA specific (14B7) and digoxigenin specific (Dig) scFv and labeled with 200nM BodipyTM conjugated fluorescent antigens. Histograms represent the mean fluorescence intensity of 10,000 E. Coli events.
  • FIG. 7 A Fluorescence distribution of ABLECTM cells expressing PA specific (14B7) and digoxigenin specific (Dig) scFv and labeled with 200nM BodipyTM conjugated fluorescent antigens. Histograms represent the
  • FIG. 7B Histograms of cells expressing 14B7 or Dig scFv labeled with 200nM of the 240kDa digoxigenin- phycoerythrin conjugate.
  • FIG. 8 Analysis of anti-PA antibody fragments selected using APEx
  • FIG. 8 A Signal Plasmon Resonance (SPR) analysis of anti-PA scAb binding to PA.
  • FIG. 8B Table of affinity data acquired by SPR.
  • FIG. 8C FC Histogram of anti-PA scFv in pAPExl expressed in E. coli and labeled with 200nM PA-BodipyTM conjugate as compared with anti- methamphetamine (Meth) scFv negative control.
  • FIG. 9 N-Terminal vs. C-Terminal anchoring strategy comparison.
  • FIG. 9 A Anti- digoxigenin Dig scfv, anti-PA Ml 8 scFv and anti-methamphetamine Meth scFv expressed as N-terminal fusions in the pAPExl vector in E. coli specifically label with 200nM of their respective antigen.
  • FIG. 9B C-terminal fusions of same scFv in pAK200 vector specifically labeled with 200nM of their respective antigen.
  • FIG. 10 View from the top of the antibody binding pocket showing the conformation and amino acid substitutions in the 1H, M5, M6 and Ml 8 sequences.
  • FIG. 9 A Anti- digoxigenin Dig scfv, anti-PA Ml 8 scFv and anti-methamphetamine Meth scFv expressed as N-terminal fusions in the pAPExl vector in E. coli specifically label with 200nM of
  • FIG. 11 Alignment of 14B7 scFv (SEQ ID ⁇ O:21) and Ml 8 scFv (SEQ ID NO:23) sequences showing variable heavy and variable light chains and mutations made to improve binding affinity.
  • FIG. 12 (FIG.12 A) Simple structure of MalF-fused expression system and (FIG.12B) fusion points in MalF protein.
  • FIG. 13 Flow cytometric analysis of MalF-fused clones (C, cytoplasmic location; P, periplasmic location of fusion points).
  • FIG. 12 (FIG.12 A) Simple structure of MalF-fused expression system and (FIG.12B) fusion points in MalF protein.
  • FIG. 13 Flow cytometric analysis of MalF-fused clones (C, cytoplasmic location; P, periplasmic location of fusion points).
  • FIG. 14 Library construction of truncated TatC fused 26-10 scFv/GFP by THIO- ITCHY method (K, Kpnl; B, Bglll; E, EcoRI; S, Smal; His6, histidine hexamer tag; tlpp, lpp terminator)
  • FIG. 15 TatC-topology prediction based on the GFP fusion clones. Dark background symbols represent the fusion points identified by sequencing. Among these, square-shaped symbols designate the fusion points of clones represented twice, diamond-shaped symbols designate clones represented three times, and pentagonal shaped symbol designate clones represented four times.
  • FIG. 16 TatC-topology prediction based on the 26-10 scFv fusion clones.
  • FIG. 17 Simple structure of the randomized NlpA library vector.
  • the invention overcomes the limitations of the prior art by providing a novel method for isolating polypeptides capable of anchoring heterologous polypeptides to the bacterial inner, membrane.
  • libraries of candidate anchor polypeptides are expressed as fusions with heterologous polypeptides capable of being detected ion the outer face of the, bacterial inner membrane.; .
  • the heterologous polypeptide is bound- to outer face of the inner membrane.
  • Bacteria with the functional anchor sequence can subsequently be identified based on the presence of the heterologous polypeptide, for example, by removing the outer membrane to remove unanchored heterologous polypeptide followed by detection of the anchored polypeptide.
  • Such bacteria may be detected in any suitable manner, including use of direct fluorescence or secondary antibodies. Fluorescent labeling allows implementation of efficient techniques such as fluorescence activated cell sorting (FACS). Identification of additional anchors is desirable in that particular anchors may function more efficiently with certain polypeptides being bound to the inner membrane due to, for example, stearic or other characteristics of the polypeptide relative to the anchor and inner membrane.
  • FACS fluorescence activated cell sorting
  • the anchor may also affect the efficiency with which a target ligand bound to a candidate binding protein is detected.
  • the anchor may vary the degree to which an antigen on a bound target ligand is exposed for detection with a labeled antibody or other detection agent.
  • the display of heterologous proteins on microbial scaffolds has attractive applications in many different areas including vaccine development, bioremediation and protein engineering.
  • Gram negative bacteria there have been display systems designed such that, by virtue of a N or C terminal chimera fusion, proteins are displayed to the cell surface.
  • fusions to outer membrane proteins, lipoproteins, surface structural proteins and leader peptides many share the same limitations.
  • One limitation is the size of the protein which can be displayed.
  • display scaffolds can only tolerate a few hundred amino acids, which significantly limits the scope of proteins which can be displayed.
  • display implies that the protein of interest is situated such that it can interact with its environment, yet the major limitation of many of these systems is that the architecture of the outer surface of gram negative bacteria and in particular the presence of lipopolysaccharide (LPS) molecules having steric limitations that inhibit the binding of externally added ligands.
  • LPS lipopolysaccharide
  • Another limitation arises from the requirement that the displayed protein is localized on the external surface of the outer membrane.
  • the polypeptide must first be secreted across the cytoplasmic membrane must then transverse the periplasmic space and finally it must be assemble properly in the outer membrane.
  • a heterologous polypeptide may be any type of detectable molecule.
  • the limitations of the prior techniques can be overcome by the identification and use of anchor polypeptides to display proteins anchored to the outer surface of the inner membrane. It was demonstrated using the technique that, by ⁇ > utilizing conditions that permeabilize the outer membrane, E. coli expressing inner membrane anchored scFv antibodies (approx. 30kDa in size) can be labeled with a target antigen conjugated, for example, to a fluorophore and can subsequently be used to sort protein libraries utilizing flow cytometry for isolation of gain of function mutants.
  • labeled antigens with sizes up to at least 240 kDa can be used to detect anchored polypeptides.
  • cells comprising functional anchors may be isolated by flow cytometry and the DNA of isolated clones rescued by PCR.
  • anchored molecules are labeled with fluorescent dyes.
  • bacterial clones expressing polypeptides fused to a functional inner membrane anchor are labeled with a fluorescently labeled molecule having specific affinity for the polypeptide on the face of the bacterial inner membrane.
  • the term "specific affinity" refers to an association that is specific to a particular set of molecules and not general to, for example, all proteins within a cell.
  • An example of specific affinity is the relationship between an antibody or fragment thereof and a given antigen.
  • the polypeptide bound on the inner membrane may itself be a ligand and/or a binding protein.
  • the fluorescent bacteria expressing functional anchors can then be enriched from the population using automated techniques such as flow cytometry, including FACS.
  • Candidate anchor sequences screened in accordance with the invention may be selected for likely function as an inner membrane protein or may be random sequences. Benefit may be obtained by screening of transmembrane or polytropic membrane polypeptides and fragments thereof.
  • an inner membrane anchor polypeptide refers to any peptide sequence that causes a polypeptide to associate with and become bound to the outer face of the inner membrane.
  • the inner membrane anchor polypeptide need not permanently bind to the inner membrane, but the association should be sufficiently strong to allow removal of the outer membrane while maintaining anchoring on the outer face of the inner membrane.
  • the periplasm comprises the space defined by the inner and outer membranes of a Gram-negative bacterium. In wild-type E. coli and other Gram negative bacteria, the outer membrane serves as a permeability barrier that severely restricts the diffusion of molecules greater than 600 Da into the periplasmic space (Decad and Nikado, 1976).
  • the inventors have identified techniques that allow fluorescent conjugates of ligands and polypeptides to pass the outer membrane and bind to proteins and remain bound to the inner membrane. Therefore, in bacterial cells expressing functional anchors, the heterologous polypeptide can be detected, allowing the bacteria to be isolated from the rest of the library.
  • heterologous it is understood that a given polypeptide need not be from a source other than the host cell, but will be in a genetic arrangement other than that of the wild type host genome.
  • cells may efficiently be isolated by flow cytometry (fluorescence activated cell sorting (FACS)).
  • FACS fluorescence activated cell sorting
  • the periplasmic compartment is contained between the inner and outer membranes of Gram negative cells (see, e.g., Oliver, 1996). As a sub-cellular compartment, it is subject to variations in size, shape and content that accompany the growth and division of the cell.
  • Within a framework of peptidoglycan heteropolymer is a dense milieu of periplasmic proteins and little water, lending a gel-like consistency to the compartment (Hobot et al, 1984; van Wielink and Duine, 1990).
  • the peptidoglycan is polymerized to different extents depending on the proximity to the outer membrane, close-up it forms the murein sacculus that affords cell shape and resistance to osmotic lysis.
  • the outer membrane (see Nikaido, 1996) is composed of phospholipids, porin proteins and, extending into the medium, lipopolysaccharide (LPS).
  • LPS lipopolysaccharide
  • the molecular basis of outer membrane integrity resides with LPS ability to bind divalent cations (Mg2+ and Ca2+) and link each other electrostatically to form a highly ordered quasi-crystalline ordered "tiled roof on the surface (Labischinski et al, 1985).
  • the membrane forms a very strict permeability barrier of allowing passage of molecules no greater than around 650 Da (Burman et al, 1972; Decad and Nikaido, 1976) via the porins.
  • the large water filled porin channels are primarily responsible for allowing free passage of mono and disaccharides, ions and amino acids in to the periplasm compartment (Naeke, 1976; Nikaido and Nakae, 1979; Nikaido and Vaara, 1985).
  • detection agents can diffuse into the periplasm. Such diffusion can be aided by one or more treatments of a bacterial cell, thereby rendering the outer membrane more permeable as is described herein.
  • polypeptide includes antibodies, fragments of antibodies, as well as any other peptides, including proteins potentially capable of binding a given target molecule.
  • the antibody or other binding peptides may be expressed with the invention as fusion polypeptides with polypeptides capable of serving as anchors to the periplasmic face of the inner membrane.
  • Such a technique may be termed "Anchored Periplasmic Expression” or "APEx”.
  • the outer membrane forms a very strict permeability barrier allowing passage of molecules no greater than around 650 Da (Burman et al, 1972; Decad and Nikaido, 1976) via the porins.
  • ligands greater than 2000 Da in size can diffuse into the periplasm without disruption of the periplasmic membrane. Such diffusion can be aided by one or more treatments of a bacterial cell, thereby rendering the outer membrane more permeable, or by removal of the outer membrane as described below.
  • a fusion polypeptide comprising a candidate anchor sequence may further comprise any second detectable polypeptide. It will be preferable that the second polypeptide is readily detectable when anchored to the outer face of the inner, membrane.
  • the polypeptide may comprise a label that is directly detectable, such , as a fluorophore including GFP, or with secondarily detectable agents such as an antigen or binding protein that can be detected by contacting with labeled binding protein or antigen.
  • the fusion may comprise a linker polypeptide serving to link the candidate anchor polypeptide and the second polypeptide.
  • a general scheme behind the invention comprises the advantageous expression of a heterogeneous collection of candidate anchors to identify functional anchor polypeptides having desirable characteristics for the display of various molecules on the outer face of the inner membrane.
  • the candidate anchor may comprise a random sequence or may be selected for likely ability to serve as an anchor sequence.
  • An example of potential sequences for use as candidate anchors include lipoproteins from E. coli and other Gram negative bacteria.
  • candidate anchor sequences may therefore comprise a periplasm sec signal sequence followed by the sec cleavage region and coding for cysteine as amino acid 1 and arginine as amino acid 2 of the mature protein.
  • Such a sequence may further be attached to other candidate sequences for screening.
  • Transmembrane proteins and fragments thereof could also potentially be used as candidate anchors, although this may require a larger fusion construct.
  • sequences for use as candidate anchors include fragments, mutants and full length sequences of lipoproteins such as Pullulanase of K. pneumoniae, phage encoded celB, and E.
  • coli acrE envC
  • inner membrane proteins such as AraH, MglC, Mal-F, MalG, Mai C, MalD, RbsC, RbsC, ArtM, ArtQ, GlnP, ProW, HisM, HisQ, LivH, LivM, LivA, Liv E,Dp ⁇ B, DppC, OppB,AmiC, AmiD, BtuC, FhuB, FecC, FecD,FecR, FepD, NikB, NikC, CysT, CysW, UgpA, UgpE, PstA, PstC, PotB, PotC,PotH, Potl, ModB, NosY, PhnM, LacY, SecY, TolC, DsbB, DsbD, TonB, TatC, CheY, TraB, Exb D, ExbB and Aas.
  • Single transmembrane loops of any cytoplasmic protein can also be used as a candidate membrane anchor.
  • the preparation of diverse populations of fusion proteins in the context of phage display is known (see, e.g., U.S. Patent 5,571,698). Similar techniques may be employed with the instant invention to prepare candidate inner membrane anchor sequences. Such fusions can be mutated to form a library of structurally related fusion proteins that can be screened for function as an anchor in accordance with the invention.
  • techniques for the creation of heterogeneous collections of candidate, molecuies which are, well known to those of skill in the art in conjunction with phage display, can be adapted for use with the invention.
  • fusion proteins comprising candidate anchor sequences, including promoters, enhancers and leader sequences.
  • the current invention provides the advantage relative to phage display of not requiring the use of phage or expression of molecules on the outer cell surface, which may be poorly expressed or may be deleterious to the host cell.
  • Methods for creation of fusion proteins are well known to those of skill in the art (see, for example, U.S. Patent 5,780,279).
  • One means for doing so comprises constructing a gene fusion between a candidate anchor polypeptide and a second polypeptide by mutating a starting candidate anchor sequence, thereby generating a family of mutants.
  • Those sequences having desired levels of function as an anchor can then be selected from large populations of bacteria expressing the family of mutants. Those bacteria in which a selected anchor polypeptide is expressed, can then be isolated and the corresponding nucleic acid encoding the anchor can be cloned.
  • methods are employed for increasing the permeability of the outer membrane to one or more detection agent(s) and/or removing the outer membrane. This can be used to improve detection of polypeptides on the face of the inner membrane indicating function of a given inner membrane anchor sequence.
  • Candidate anchor sequences may comprise large libraries of diverse candidate substances, or, alternatively, may comprise particular classes of sequences selected with an eye towards structural attributes that are believed to make them more likely to function as an inner membrane anchor.
  • a candidate molecule capable of serving as an anchor in accordance with the invention, one may carry out the steps of: providing a population of Gram negative bacterial cells comprising fusion proteins between candidate anchor sequences and a heterologous polypeptide and allowing the fusion protein to be expressed; removing the outer membrane to remove unanchored polypeptide; and detecting the presence of the heterologous polypeptide on the surface of the inner membrane of bacteria expressing a functional anchor sequence.
  • the anchor can then be cloned from the bacteria.
  • the function of an anchor will prevent diffusing out of the cell. Labeling may then be used to isolate the cell expressing a sequence serving as an inner membrane anchor, and in this way, the sequence encoding the anchor polypeptide isolated.
  • the anchor may be used, for example, in techniques such as anchored periplasmic expression.
  • the term “candidate inner membrane anchor” refers to any polypeptide ⁇ .. capable of functioning as a sequence capable of anchoring heterologous polypeptides to the outer face of the bacteria inner membrane. Such a sequences may be particularly designed for the likelihood that it will function as an inner membrane anchor.
  • A. Cloning of Anchor Sequences After a bacterial cell is identified that produces an inner membrane anchor with the desired function, the corresponding coding sequence may be cloned. In this manner, DNA encoding the anchor can be isolated and sequenced using conventional procedures (e.g., by using oligonucleotide probes that are capable of binding specifically to genes encoding the anchor).
  • the anchor may be placed into expression vectors, which can then transfected into bacterial host cells for the display of polypeptides on the inner membrane.
  • the DNA may be modified, for example, by substituting codons and adding linker or leader peptides as desired. In that manner, "chimeric" or “hybrid” anchor polypeptides are prepared that have the desired function.
  • nucleic acids may be cloned from viable or non- viable cells. In the case of nonviable cells, for example, it may be desired to use amplification of the cloned DNA, for example, using PCR. This may also be carried out using viable cells either with or without further growth of cells.
  • a candidate anchor sequence may be fused to a binding protein that is detected with a labeled ligand, or alternatively, may be fused to a ligand and detected with a labeled binding protein.
  • labeled ligands and binding proteins of potentially any size may be used. In the absence of removal of the periplasmic membrane, it will typically be preferable that reagents of less than 50,000 Da in size be used in order to allow efficient diffusion of the ligand across the bacterial periplasmic membrane.
  • Labeled ligands or antibodies used to detect a given polypeptide can be prepared, for example, by linking the ligand or binding protein to at least one detectable agent to form a conjugate. For example, it is conventional to link or covalently bind or complex at least one detectable molecule or moiety.
  • a "label” or “detectable label” is a compound and/or element that can be detected due to specific functional properties, and/or chemical characteristics, the i use of which allows the compound to which it is attached to be detected, and/or further . quantified if desired.
  • a visually-detectable marker is used such that automated screening of cells for the label can be carried out.
  • fluorescent labels are beneficial in that they allow use of flow cytometry for isolation of cells expressing a desired binding protein or antibody. Examples of agents that may be detected by visualization with an appropriate instrument are known in the art, as are methods for their attachment to a desired ligand (see, e.g., U.S.
  • Such agents can include paramagnetic ions; radioactive isotopes; fluorochrom.es; NMR-detectable substances and substances for X-ray imaging.
  • Types of fluorescent labels that may be used with the invention will be well known to those of skill in the art and include, for example, Alexa 350, Alexa 430, AMCA, BODIPY 630/650, BODIPY 650/665, BODIPY-FL, BODIPY-R6G, BODIPY-TMR, BODIPY-TRX, Cascade Blue, Cy3, Cy5,6-FAM, Fluorescein Isothiocyanate, HEX, 6- JOE, Oregon Green 488, Oregon Green 500, Oregon Green 514, Pacific Blue, REG, Rhodamine Green, Rhodamine Red, Renographin, ROX, TAMRA, TET, Tetramethylrhodamine, and/or Texas Red.
  • Magnetic screening techniques are well known to those of skill in the art (see, for example, U.S. Pat. No. 4,988,618, U.S. Pat. No. 5,567,326 and U.S. Pat. No. 5,119,901).
  • paramagnetic ions that could be used as labels in accordance with such techniques include ions such as chromium (III), manganese (II), iron (III), iron (II), cobalt (II), nickel (II), copper (II), neodymium (III), samarium (III), ytterbium (El), gadolinium (III), vanadium (II), terbium (III), dysprosium (III), holmium (III) and/or erbium (III).
  • Ions useful in other contexts include but are not limited to lanthanum (III), gold (III), lead (II), and especially bismuth (III) .
  • Another type of detection reagent contemplated in the present invention are those linked to a secondary binding molecule and/or to an enzyme (an enzyme tag) that will generate a colored product upon contact with a chromogenic substrate. Examples of such enzymes include urease, alkaline phosphatase, (horseradish) hydrogen peroxidase or glucose oxidase. h such instances, it will be desired that cells selected remain viable.
  • Preferred secondary binding agents are biotin and/or avidin and streptavidin compounds.
  • 2- and 8-azido analogues of purine nucleotides have been used as site-directed photoprobes to identify nucleotide-binding proteins in crude cell extracts (Owens & Haley, 1987; Atherton et al, 1985).
  • the 2- and 8-azido nucleotides have also been used to map nucleotide-binding domains of purified proteins (Khatoon et al, 1989; King et al, 1989; and Dholakia et al, 1989) and may be used as binding agents. Labeling can be carried out by any of the techniques well known to those of skill in the art.
  • ligands and binding proteins can be labeled by contacting with the desired label and a chemical oxidizing agent such as sodium hypochlorite, or an enzymatic oxidizing agent, such as lactoperoxidase.
  • a chemical oxidizing agent such as sodium hypochlorite, or an enzymatic oxidizing agent, such as lactoperoxidase.
  • an exchange process could be used.
  • direct labeling techniques may be used, e.g., by incubating with the label, a reducing agent such as SNC1 2 , a buffer solution such as sodium-potassium phthalate solution, and the agent to be labeled.
  • Intermediary functional groups could also be used, for example, to bind labels to the detection reagent in the presence of diethylenetriaminepentaacetic acid (DTP A) or ethylene diaminetetracetic acid (EDTA).
  • DTP A diethylenetriaminepentaacetic acid
  • EDTA ethylene diaminetetracetic acid
  • Some attachment methods involve the use of an organic chelating agent such as diethylenetriaminepentaacetic acid anhydride (DTPA); ethylenetriaminetetraacetic acid; N-chloro-p-toluenesulfonamide; and/or tetrachloro-3 ⁇ -6 ⁇ - diphenylglycouril-3 attached to the ligand (U.S. Patents 4,472,509 and 4,938,948, each incorporated herein by reference).
  • DTPA diethylenetriaminepentaacetic acid anhydride
  • ethylenetriaminetetraacetic acid N-chloro-p-toluenesulfonamide
  • tetrachloro-3 ⁇ -6 ⁇ - diphenylglycouril-3 attached to the ligand
  • Compounds also may be reacted with an enzyme in the presence of a coupling agent such as glutaraldehyde or periodate.
  • Conjugates with fluorescein markers can be prepared in the presence of these coupling agents or by reaction with an isothiocyanate.
  • imaging of breast tumors is achieved using monoclonal antibodies and the detectable imaging moieties are bound to the antibody using linkers such as methyl-p-hydroxybenzimidate or N-succinimidyl-3-(4- hydroxyphenyl)propionate.
  • FACS fluorescence activated cell sorting
  • Instruments for carrying out flow cytometry are known to those of skill in the art and are commercially available to the public. Examples of such instruments include FACS Star Plus, FACScan and FACSort instruments from Becton Dickinson (Foster City, Calif) Epics C from Coulter Epics Division (Hialeah, Fla.) and MoFlo from Cytomation (Colorado Springs, Co).
  • Flow cytometric techniques in general involve the separation of cells or other particles in a liquid sample.
  • the purpose of flow cytometry is to analyze the separated particles for one or more characteristics thereof, for example, presence of a labeled ligand or other molecule.
  • the basis steps of flow cytometry involve the direction of a fluid sample through an apparatus such that a liquid stream passes through a sensing region.
  • the particles should pass one at a time by the sensor and are categorized base on size, refraction, light scattering, opacity, roughness, shape, fluorescence, etc. Rapid quantitative analysis of cells proves useful in biomedical research and medicine.
  • Apparati permit quantitative multiparameter analysis of cellular properties at rates of several thousand cells per second. These instruments provide the ability to differentiate among cell types.
  • U.S. Patent 3,826,364 an apparatus which physically separates particles, such as functionally different cell types.
  • a laser provides illumination which is focused on the stream of particles by a suitable lens or lens system so that there is highly localized scatter from the particles therein.
  • high intensity source illumination is directed onto the stream of particles for the excitation of fluorescent particles in the stream.
  • Certain particles in the stream may be selectively charged and then separated by deflecting them into designated receptacles.
  • a classic form of this separation is via fluorescent-tagged antibodies, which are used to mark one or more cell types for separation.
  • Other examples of methods for flow cytometry that could include, but are not limited to, those described in U.S. Patent Nos.
  • Nucleic acid-based expression systems may find use, in certain embodiments of the invention, for the expression of recombinant proteins. For example, one embodiment of the invention involves transformation of Gram negative bacteria with the coding sequences of fusion polypeptides comprising a detectable heterologous polypeptide fused to a plurality of candidate inner membrane anchor polypeptides. Certain aspects of the invention may therefore comprise delivery of nucleic acids to target cells.
  • bacterial host cells may be transformed with nucleic acids encoding candidate anchor sequences targeted to the inner membrane of the bacteria.
  • Suitable methods for nucleic acid delivery for transformation of a cell are believed to include virtually any method by which a nucleic acid (e.g., DNA) can be introduced into such a cell.
  • Such methods include, but are not limited to, direct delivery of DNA such as by injection (U.S. Patents 5,994,624, 5,981,274, 5,945,100, 5,780,448, 5,736,524, 5,702,932, 5,656,610, 5,589,466 and 5,580,859, each incorporated herein by reference), including microinjection (Harlan and Weintraub, 1985; U.S.
  • Patent 5,789,215 incorporated herein by reference
  • electroporation U.S. Patent 5,384,253, incorporated herein by reference
  • calcium phosphate precipitation Graham and Van,' Der Eb, 1973; Chen and Okayama, 1987; Rippe et al, 1990
  • DEAE-dextran DEAE-dextran
  • a nucleic acid is introduced into a cell via electroporation. Electroporation involves the exposure of a suspension of cells and DNA to a high-voltage electric discharge.
  • certain cell wall-degrading enzymes such as pectin-degrading enzymes
  • recipient cells can be made more susceptible to transformation by mechanical wounding.
  • a nucleic acid is introduced to the cells using calcium phosphate precipitation. Human KB cells have been transfected with adenovirus 5 DNA (Graham and Van Der Eb, 1973) using this technique.
  • mice L(A9), mouse C127, CHO, CV-1, BHK, NIH3T3 and HeLa cells were transfected with a neomycin marker gene (Chen and Okayama, 1987), and rat hepatocytes were transfected with a variety of marker genes (Rippe et al, 1990).
  • A. Vectors Vectors may find use with the current invention, for example, in the transformation of a Gram negative bacterium with a nucleic acid sequence encoding a candidate anchor polypeptide which one wishes to screen for ability to anchor a given polypeptide to the inner membrane.
  • an entire heterogeneous "library" of nucleic acid sequences encoding candidate anchor sequences may be introduced into a population of bacteria, thereby allowing screening of the entire library.
  • vector is used to refer to a carrier nucleic acid molecule into which a nucleic acid sequence can be inserted for introduction into a cell where it can be replicated.
  • a nucleic acid or polypeptide sequence can be "exogenous,” or “heterologous", which means that it is foreign to the cell into which the vector is being introduced or that the sequence is homologous to a sequence in the cell but in a position within the host cell nucleic acid in which the sequence is ordinarily not found.
  • a heterologous polypeptide may therefore be isolated from the same cell in which it is expressed, but positioned at a different location.
  • Vectors include plasmids, cosmids, viruses (bacteriophage, animal viruses, and plant viruses), and artificial chromosomes (e.g., YACs).
  • viruses bacteria, animal viruses, and plant viruses
  • artificial chromosomes e.g., YACs.
  • One of skill in the art may construct a vector through standard recombinant techniques, which are described in Maniatis et al, 1988 and Ausubel et al, 1994, both of which references are incorporated herein by reference.
  • expression vector refers to a vector containing a nucleic acid sequence coding for at least part of a gene product capable of being transcribed.
  • RNA molecules are then translated into a protein, polypeptide, or peptide. In other cases, these sequences are not translated, for example, in the production of antisense molecules or ribozymes.
  • Expression vectors can contain a variety of "control sequences,” which refer to nucleic acid sequences necessary for the transcription and possibly translation of an operably linked coding sequence in a particular host organism. In addition to control sequences that govern transcription and translation, vectors and expression vectors may contain nucleic acid sequences that serve other functions as well and are described infra. 1. Promoters and Enhancers A "promoter" is a control sequence that is a region of a nucleic acid sequence at which initiation and rate of transcription are controlled.
  • RNA polymerase may contain genetic elements at which regulatory proteins and molecules may bind such as RNA polymerase and other transcription factors.
  • the phrases "operatively positioned,” “operatively linked,” “under control,” and “under transcriptional control” mean that a promoter is in a correct functional location and/or orientation in relation to a nucleic acid sequence to control transcriptional initiation and/or expression of that sequence.
  • a promoter may or may not be used in conjunction with an "enhancer,” which refers to a cis-acting regulatory sequence involved in the transcriptional activation of a nucleic acid sequence.
  • a promoter may be one naturally associated with a gene or sequence, as may be obtained by isolating the 5' non-coding sequences located upstream of the coding segment and/or exon.
  • an enhancer may be one naturally associated with a nucleic acid sequence, located either downstream or upstream of that sequence.
  • an enhancer may be one naturally associated with a nucleic acid sequence, located either downstream or upstream of that sequence.
  • certain advantages will be gained by positioning the coding nucleic acid segment under the control of a recombinant or heterologous promoter, which refers to a promoter that is not normally associated with a nucleic acid sequence in its natural environment.
  • a recombinant or heterologous enhancer refers also to an enhancer not normally associated with a nucleic acid sequence in its natural environment.
  • Such promoters or enhancers may include promoters or enhancers of other genes, and promoters or enhancers isolated from any other prokaryotic, viral, or eukaryotic cell, and promoters or enhancers not "naturally occurring," i.e., containing different elements of different transcriptional regulatory regions, and/or mutations that alter expression.
  • sequences may be produced using recombinant cloning and/or nucleic acid amplification technology, including PCR, in connection with the compositions disclosed herein (see U.S. Patent 4,683,202, U.S. Patent 5,928,906, each incorporated herein by reference).
  • control sequences that direct transcription and/or expression of sequences within non-nuclear organelles such as mitochondria, chloroplasts, and the like, can be employed as well.
  • a promoter and/or enhancer that effectively directs the expression of the DNA segment in the cell type, organelle, and organism chosen for expression.
  • One example of such promoter that may be used with the invention is the E. coli arabinose promoter.
  • the promoters employed may be constitutive, tissue-specific, inducible, and/or useful under the appropriate conditions to direct high level expression of the introduced DNA segment, such as is advantageous in the large-scale production of recombinant proteins and/or peptides.
  • the promoter may be heterologous or endogenous.
  • a specific initiation signal also may be required for efficient translation of coding sequences. These signals include the ATG initiation codon or adjacent sequences. Exogenous translational control signals, including the ATG initiation codon, may need to be provided.
  • One of ordinary skill in the art would readily be capable of determining this and providing the necessary signals.
  • MCS multiple cloning site
  • Restriction enzyme digestion refers to catalytic cleavage of a nucleic acid molecule with an enzyme that functions only at specific locations in a nucleic acid molecule. Many of these restriction enzymes are commercially available. Use of such enzymes is understood by those of skill in the art. Frequently, a vector is linearized or fragmented using a restriction enzyme that cuts within the MCS to enable exogenous sequences to be ligated to the vector. "Ligation” refers to the process of forming phosphodiester bonds between two nucleic acid fragments, which may or may not be contiguous with each other. Techniques involving restriction enzymes and ligation reactions are well known to those of skill in the art of recombinant technology. 4.
  • Termination Signals The vectors or constructs prepared in accordance with the present invention will generally comprise at least one termination signal.
  • a “termination signal” or “terminator” is comprised of the DNA sequences involved in specific termination of an RNA transcript by an RNA polymerase. Thus, in certain embodiments, a termination signal that ends the production of an RNA transcript is contemplated.
  • a terminator may be necessary in vivo to achieve desirable message levels. Terminators contemplated for use in the invention include any known terminator of transcription described herein or known to one of ordinary skill in the art, including but not limited to, for example, rhp dependent or rho independent terminators.
  • the termination signal may be a lack of transcribable or translatable sequence, such as due to a sequence truncation.
  • Origins of Replication In order to propagate a vector in a host cell, it may contain one or more origins of replication sites (often termed "ori"), which is a specific nucleic acid sequence at which replication is initiated. Alternatively an autonomously replicating sequence (ARS) can be employed if the host cell is yeast. 6. Selectable and Screenable Markers In certain embodiments of the invention, cells containing a nucleic acid construct of the present invention may be identified in vitro or in vivo by including a marker in the expression vector. Such markers would confer an identifiable change to the cell permitting easy identification of cells containing the expression vector.
  • a selectable marker is one that confers a property that allows for selection.
  • a positive selectable marker is one in which the presence of the marker allows for its selection, while a negative selectable marker is one in which its presence prevents its selection.
  • An example of a positive selectable marker is a drug resistance marker.
  • a drug selection marker aids in the cloning and identification of transformants, for example, genes that confer resistance to neomycin, puromycin, hygromycin, DHFR, GPT, zeocin and histidinol are useful selectable markers.
  • markers conferring a phenotype that allows for the discrimination of transformants based on the implementation of conditions are also contemplated.
  • screenable enzymes such as herpes simplex virus thymidine kinase (tk) or chloramphenicol acetyltransferase (CAT) may be utilized.
  • tk herpes simplex virus thymidine kinase
  • CAT chloramphenicol acetyltransferase
  • One of skill in the art would also know how to employ immunologic markers, possibly in conjunction with FACS analysis. The marker used is not believed to be important, so long as it is capable of being expressed simultaneously with the nucleic acid encoding a gene product. Further examples of selectable and screenable markers are well known to one of skill in the art. B.
  • host cell refers to a prokaryotic cell, and it includes any transformable organism that is capable of replicating a vector and/or expressing a heterologous gene encoded by a vector.
  • a host cell can, and has been, used as a recipient for vectors.
  • a host cell may be "transfected” or “transformed,” which refers to a process by which exogenous nucleic acid is transferred or introduced into the host cell.
  • a transformed cell includes the primary subject cell and its progeny.
  • a host cell is a Gram negative bacterial cell. These bacteria are suited for use with the invention in that they posses a periplasmic space between the inner and outer membrane and, particularly, the aforementioned inner membrane between the periplasm and cytoplasm, which is also known as the cytoplasmic ' membrane. :As such, any other cell with ' such a periplasmic space could be used in accordance with the invention. Examples of, Gram negative bacteria that may find use with the invention may include, but are not limited to, E.
  • the Gram negative bacterial cell may be still further defined as bacterial cell which has been transformed with the coding sequence of a fusion polypeptide comprising a candidate binding polypeptide capable of binding a selected ligand.
  • the polypeptide is anchored to the outer face of the cytoplasmic membrane, facing the periplasmic space, and may comprise an antibody coding sequence or another sequence.
  • One means for expression of the polypeptide is by attaching a leader sequence to the polypeptide capable of causing such directing.
  • a leader sequence to the polypeptide capable of causing such directing.
  • Numerous prokaryotic cell lines and cultures are available for use as a host cell, and they can be obtained through the American Type Culture Collection (ATCC), which is an organization that serves as an archive for living cultures and genetic materials (www.atcc.org).
  • ATCC American Type Culture Collection
  • An appropriate host can be determined by one of skill in the art based on the vector backbone and the desired result.
  • a plasmid or cosmid for example, can be introduced into a prokaryote host cell for replication of many vectors.
  • Bacterial cells used as host cells for vector replication and/or expression include DH5 ⁇ , JM109, and KC8, as well as a number of commercially available bacterial hosts such as SURE ® Competent Cells and SOLOPACKTM Gold Cells (STRATAGENE ® , La Jolla).
  • bacterial cells such as E. coli LE392 could be used as host cells for bacteriophage.
  • Many host cells from various cell types and organisms are available and would be known to one of skill in the art.
  • a viral vector may be used in conjunction with a prokaryotic host cell, particularly one that is permissive for replication or expression of the vector.
  • Some vectors may employ control sequences that allow it to be replicated and/or expressed in both prokaryotic and eukaryotic cells.
  • One of skill in the art would further understand the conditions under which to incubate all of the above described host cells to maintain them and to permit replication of a vector.
  • Also understood and known are techniques and conditions that would allow large-scale production of vectors, as well as production of the nucleic acids encoded by vectors and their cognate polypeptides, proteins, or peptides.
  • C. Expression Systems Numerous expression systems exist that comprise at least a part or all of the compositions discussed above. Such systems could be used, for example, for the production of a polypeptide product identified in accordance with the invention as capable of anchoring a particular polypeptide on the inner membrane.
  • Prokaryote- -based systems can be employed for use with the present invention to produce nucleic acid sequences, or their cognate polypeptides, proteins and peptides. Many such systems are commercially and widely available.
  • Other examples of expression systems comprise of vectors containing a strong prokaryotic promoter such as T7, Tac, Trc, BAD, lambda pL, Tetracycline or Lac promoters, the pET Expression System and an E. coli expression system.
  • nucleic acids used as a template for amplification may be isolated from cells, tissues or other samples according to standard methodologies (Sambrook et al, 1989). In certain embodiments, analysis may be performed on whole cell or tissue homogenates or biological fluid samples without substantial purification of the template nucleic acid.
  • the nucleic acid may be genomic DNA or fractionated or whole cell RNA. Where RNA is used, it may be desired to first convert the RNA to a complementary DNA.
  • primer as used herein, is meant to encompass any nucleic acid that is capable of priming the synthesis of a nascent nucleic acid in a template-dependent process.
  • primers are oligonucleotides from ten to twenty and/or thirty base pairs in length, but longer sequences can be employed. Primers may be provided in double-stranded and/or single-stranded form, although the single-stranded form is preferred. Pairs of primers designed to selectively hybridize to nucleic acids corresponding to a selected nucleic acid sequence are contacted with the template nucleic acid under conditions that permit selective hybridization. Depending upon the desired application, high stringency hybridization conditions may be selected that will only allow hybridization to sequences that are completely complementary to the primers. In other embodiments, hybridization may occur under reduced stringency to allow for amplification of nucleic acids contain one or more mismatches with the primer sequences.
  • the template-primer complex is contacted with one or more enzymes that facilitate template-dependent nucleic acid synthesis.
  • Multiple rounds of amplification also referred to as "cycles," are conducted until a sufficient amount of amplification product is produced.
  • the amplification product may be detected or quantified. In certain applications, the detection may be performed by visual means. Alternatively, the detection may involve indirect identification of the product via chemiluminescence, radioactive scintigraphy of incorporated radiolabel or fluorescent label or even via a system using electrical and/or thermal impulse signals (Affymax technology; Bellus, 1994).
  • a number of template dependent processes are available to amplify the oligonucleotide sequences present in a given template sample.
  • PCR polymerase chain reaction
  • a reverse transcriptase PCR amplification procedure may be performed to quantify the amount of mRNA amplified.
  • Methods of reverse transcribing RNA into cDNA are well known (see Sambrook et al, 1989).
  • Alternative methods for reverse transcription utilize thermostable DNA polymerases. These methods are described in WO 90/07641.
  • Polymerase chain reaction methodologies are well known in the art. Representative methods of RT-PCR are described in U.S. Patent 5,882,864.
  • Another method for amphfication is ligase chain reaction ("LCR”), disclosed in European Application 320 308, incorporated herein by reference in its entirety.
  • LCR ligase chain reaction
  • 4,883,750 describes a method similar to LCR for binding probe pairs to a target sequence.
  • PCT/US87/00880 may also be used as an amplification method in the present invention.
  • a replicative sequence of RNA that has a region complementary to that of a target is added to a sample in the presence of an RNA polymerase.
  • the polymerase will copy the replicative sequence which may then be detected.
  • An isothermal amplification method in which restriction endonucleases and ligases are used to achieve the amplification of target molecules that contain nucleotide 5'-[alpha-thio] ⁇ triphosphates in one strand of a restriction site may also be useful in the amplification of nucleic acids in the present invention (Walker et al, 1992). Strand Displacement Amplification (SDA), disclosed in U.S.
  • SDA Strand Displacement Amplification
  • Patent 5,916,779 is another method of carrying out isothermal amplification of nucleic acids which involves multiple rounds of strand displacement and synthesis, i.e., nick translation.
  • Other nucleic acid amplification procedures include transcription-based amplification systems (TAS), including nucleic acid sequence based amplification (NASBA) and 3SR (Kwoh et al, 1989; Gingeras et al, PCT Apphcation WO 88/10315, incorporated herein by reference in their entirety).
  • TAS transcription-based amplification systems
  • NASBA nucleic acid sequence based amplification
  • 3SR Zaoh et al, 1989; Gingeras et al, PCT Apphcation WO 88/10315, incorporated herein by reference in their entirety.
  • 329 822 disclose a nucleic acid amplification process involving cyclically synthesizing single-stranded RNA (“ssRNA”), ssDNA, and double-stranded DNA (dsDNA), which may be used in accordance with the present invention.
  • ssRNA single-stranded RNA
  • dsDNA double-stranded DNA
  • PCT Application WO 89/06700 discloses a nucleic acid sequence amphfication scheme based on the hybridization of a promoter region/primer sequence to a target single-stranded DNA (“ssDNA”) followed by transcription of many RNA copies of the sequence. This scheme is not cyclic, i.e., new templates are not produced from the resultant RNA transcripts.
  • Other amplification methods include "race” and "one-sided PCR” (Frohman, 1990; Ohara et ⁇ /., 1989).
  • EXAMPLE 1 Demonstration of Anchored Periplasmic Expression to Target Small Molecules and Peptides
  • the ability of scFvs displayed by APEx to target small molecules and peptides is shown in FIGs. 1A-1B and in FIG. IC, respectively.
  • Three cultures of Escherichia coli containing fusions of the first six amino acids of NlpA (to serve as a inner membrane targeting sequence for APEx analysis) to either an anti-methamphetamine, anti-digoxin, or anti-peptide scfv were grown up and induced for protein expression as described below. Cells of each construct were then labeled in 5xPBS buffer with 200nM concentrations of methamphetamine-FL (FIG.
  • EXAMPLE 2 Demonstration of Recognition of Ab Fragments by Anchored Periplasmic Expression
  • polyclonal antibody serum against human Ab fragments or mouse Ab fragments would recognize scFvs derived from each displayed on the E. coli inner membrane by anchored periplasmic expression.
  • Cy5 antigen conjugated fluorophore as compared to the cells expressing the an anti- digoxigenin scFv (FIG. 3A).
  • digoxigenin was coupled to phycoerythrin(PE), a 240kDa fluorescent protein. Cells were labeled with this conjugate as described below. It was found that E. coli (10,000 events) expressing the anti-digoxigenin scFv via anchored periplasmic expression were labeled with the large PE-digoxigenin conjugate while those expressing a non-specific scFv via anchored periplasmic expression show little fluorescence (FIG. 3B).
  • EXAMPLE 4 Demonstration of Selecting for Improved scFv Variants from a Library of scFvs by Flow Cytometric Selection. Scans were carried out of polyclonal Escherichia coli expressing, via anchored periplasmic expression, a mutagenic library of an scFv with affinity to methamphetamine. Through two rounds of sorting and re-sorting using a Methamphetamine conjugated fluorophore, a sub-population of the library was isolated. (FIG. 4) Individual clones from this library were labeled with the same Methamphetamine flourophore and analyzed as described below. Shown in FIG. 5 is an example of a clone, designated mutant 9, that had a higher mean FL signal than the parent anti-methamphetamine scFv.
  • EXAMPLE 5 Materials and Methods: A. Vector Construction The leader peptide and first six amino acids of the mature NlpA protein were generated by whole cell PCR (Perken Elmer) on XLl-blue Escherichia coli, (Stratagene) using primers BRH#08 5' GAAGGAGATATACATATGAAACTGACAACACATCATCTA 3' (SEQ • ID NO:6) and BRH#9 5' CTGGGCCATGGCCGGCTGGGCCTCGCTGCTACTCTGGTCGCAACC 3', (SEQ ID NO:7) VENT polymerase (New England Biolabs) and dNTPs (Roche).
  • coli strains TGI and HB2151 were provided with the Griffin library.
  • ABL ⁇ TMC and ABLETMK were purchased from Stratagene and helper phage M13K07 from Pharmacia.
  • a positive control for FACS analysis of a phage display vehicle was constructed by replacing a pre-existing scFv in pHEN2 with the 26.10 scFv to create pHEN2.dig.
  • the negative control was pHEN2.thy bearing the anti-thyroglobulin scFv provided with the Griffin.1 library.
  • the P tao vector was a derivative of pIMS120 (Hayhurst, 2000).
  • the Griffin.1 library is a semi-synthetic scFv library derived from a large repertoire of human heavy "and light chains with part or all of the CDR3 loops randomly mutated and recombined in vivo (Griffiths et al, 1994).
  • the library represents one potential source of candidate binding polypeptides for screening by anchored periplasmic expression in accordance with the invention.
  • the library was rescued and subjected to five rounds of panning according to the web-site instruction manual (www.mrc- cpe.cam.ac.uk/ ⁇ phage/glp.html).
  • hnmunotubes were coated with lO ⁇ gml "1 digoxin-BSA conjugate and the neutralized eluates were halved and used to infect either TG-1 for the next round of phage panning, or ABLETM C for FACS analysis. Eluate titers were monitored to indicate enrichment of antigen binding phage. To confirm reactivity, a polyclonal phage ELISA of purified, titer normalized phage stocks arising from each round was performed on digoxin-ovalbumin conjugate. The percentage of positive clones arising in rounds 3, 4 and 5 was established by monoclonal phage ELISA of 96 isolates after each round.
  • a positive was arbitrarily defined as an absorbance greater than 0.5 with a background signal rarely above 0.01. Mval fingerprinting was applied to 24 positive clones from rounds 3, 4 and 5.
  • F. FACS screening For scanning with APEx expression, glycerol stocks of E. coli carrying the APEx construct were grown and labeled as described in section B and C. Following labeling cells were washed once in PBS and scanned. In the aforementioned studies using bodipy or FL labeled antigen, a 488nm laser for excitation was used, while with Cy5 a 633nm laser was used.
  • Sorting with APEx expression was as follows: all sorts were performed using a MoFlo FC (Cytomation). Previously described libraries were grown and labeled as described in section B and C, washed once with PBS and sorted for increased FL intensity. Subsequent rounds of sorting were applied until polyclonal scans of the population demonstrate enrichment. (See FIG. 4) Individual clones were then picked and analyzed for FL activity.
  • phage particles by ELISA Screening phage particles by ELISA is summarized as follows. Binding of phage in ELISA is detected by primary sheep anti-M13 antisera (CP laboratories or 5 prime - 3 prime) followed by a horseradish peroxidase (HRP) conjugated anti-sheep antibody (Sigma). Alternatively, a HRP-anti-M13 conjugate can be used (Pharmacia). Plates can be blocked with 2% MPBS or 3% BSA-PBS. For the polyclonal phage ELISA, the technique is generally as follows: coat MicroTest III flexible assay plates (Falcon) with 100 ⁇ l per well of protein antigen.
  • Falcon MicroTest III flexible assay plates
  • Antigen is normally coated overnight at 4°C at a concentration of 10-100 ⁇ g/ml in either PBS or 50 mM sodium hydrogen carbonate, pH 9.6. Rinse wells 3 times with PBS, by flipping over the ELISA plates to discard excess liquid, and fill well with 2% MPBS or 3% BSA-PBS for 2 hr at 37°C. Rinse wells 3 times with PBS. Add 10 ⁇ l PEG precipitated phage from the stored aliquot of phage from the end of each round of selection (about 10 10 tfu.). Make up to 100 ⁇ l with 2% MPBS or 3% BSA-PBS. Incubate for 90 min at rt.
  • the pHEN phage particles need to be rescued: Inoculate individual colonies from the plates in CIO (after each round of selection) into 100 ⁇ l 2xTY containing 100 ⁇ g/ml ampicillin and 1 % glucose in 96-well plates (Corning 'Cell Wells') and grow with shaking (300 rpm.) overnight at 30°C. Use a 96-well transfer device to transfer a small inoculum (about 2 ⁇ l) from this plate to a second 96-well plate containing 200 ⁇ l of 2xTY containing 100 ⁇ g/ml ampicillin and 1% glucose per well. Grow shaking at 37°C for 1 hr.
  • the selected pHEN needs to be infected into HB2151 and then induced to give soluble expression of antibody fragments for ELISA. From each selection take 10 ⁇ l of eluted phage (about 10 5 t.u.) and infect 200 ⁇ l exponentially growing HB2151 bacteria for 30 min at 37°C (waterbath). Plate 1, 10, 100 ⁇ l, and 1:10 dilution on TYE containing 100 ⁇ g/ml ampicillin and 1% glucose. Incubate these plates overnight at 37°C.
  • LMB3 CAG GAA ACA GCT ATG AC (SEQ ID NO:l) and Fd seql: GAA TTT TCT GTA TGA GG (SEQ ID NO:2).
  • Fd seql GAA TTT TCT GTA TGA GG
  • SEQ ID NO:3 For sequencing of the VH and VL, use is recommend of the primers FOR_LinkSeq: GCC ACC TCC GCC TGA ACC (SEQ ID NO:3) and pHEN-SEQ: CTA TGC GGC CCC ATT CA (SEQ ID NO:4).
  • FC flow cytometry
  • Bacterial and yeast protein display in combination with FC has been employed for the engineering of high affinity antibodies to a variety of ligands (Daugherty et al, 1999; Boder et al, 2000).
  • the requirement for the display of proteins on cell surfaces imposes a number of biological constraints that can impact library screening applications.
  • Processes such as the unfolded protein response in eucaryotes or the stringency of protein sorting to the outer membrane of Gram-negative bacteria limit the diversity of the polypeptides that are actually compatible with surface display (Sagt et al, 2002; Sathopoulos et al, 1996).
  • microbial surfaces are chemically complex structures whose macromolecular composition can interfere with proteimligand recognition.
  • APEx overcomes the biological constraints and antigen access limitations of previous display strategies, enabling the efficient isolation of antibodies to virtually any size antigen.
  • proteins are tethered to the external (periplasmic) side of the E. coli cytoplasmic membrane as either N- or C-terminal fusions, thus eliminating biological constraints associated with the display of proteins on the cell surface.
  • coli cells expressing anchored scFv antibodies can be specifically labeled with fluorescent antigens, of at least 240 kDa, and analyzed by FC.
  • fluorescent antigens of at least 240 kDa
  • FC fluorescent antigen
  • the inventors have demonstrated the efficient isolation of antibodies with markedly improved ligand affinities, including an antibody fragment to the protective antigen of Bacillus anthracis with an affinity that was increased over 120-fold.
  • A. Anchored Periplasmic Expression and Detection of Ligand Binding For screening applications, an ideal expression system should minimize cell toxicity or growth abnormalities that can arise from the synthesis of heterologous polypeptides (Daugherty et al, 2000).
  • APEx avoids the complications that are associated with transmembrane protein fusions (Miroux and Walker, 1996; Mingarro et al, 1997). Unlike membrane proteins, bacterial lipoproteins are not known to require the SRP or YidC pathways for membrane anchoring (Samuelson et al, 2000). Lipoproteins are secreted across the membrane via the Sec pathway and once in the periplasm, a diacylglyceride group is attached through a thioether bond to a cysteine residue on the C-terminal side of the signal sequence.
  • the signal peptide is then cleaved by signal peptidase II, the protein is fatty acylated at the modified cysteine residue, and finally the lipophilic fatty acid inserts into the membrane, thereby anchoring the protein (Pugsley, 1993; Seydel et al, 1999; Yajushi et al, 2000). . .
  • a sequence encoding the leader peptide and first six amino acids of the mature NlpA (containing the putative fatty acylation and inner membrane targeting sites) was employed for anchoring scFv antibodies to the periplasmic face of the inner membrane.
  • NlpA is a non- essential E.
  • the cells were incubated with EDTA and lysozyme to disrupt the outer membrane and the cell wall.
  • the permeabilized cells were mixed with the respective antigens conjugated to the fluorescent dye BODIPYTM (200 nM) and the cell fluorescence was determined by flow cytometry.
  • Treated cells expressing the NlpA-[14B7 scFv] and the NlpA- [Dig scFv] exhibited an approximate 9-fold and 16-fold higher mean fluorescence intensity, respectively, compared to controls (FIG. 7A).
  • scFvs anchored on the cytoplasmic membrane can readily bind to ligands ranging from small molecules to proteins of at least up to 240 kDa in molecular weight.
  • labeling with digoxigenin-PE followed by one round of flow cytometry resulted in an over 500-fold enrichment of bacteria expressing NlpA-[Dig scFv] from cells expressing a similar fusion with a scFv having unrelated antigen specificity.
  • MoFlo droplet deflection flow cytometer selectively gating for low PI fluorescence (630 nm emission) and high BODIPYTM fluorescence. Approximately 5% of the cells sorted with the highest 530nm fluorescence (FL1) were collected, immediately restained with PI alone and resorted as above. Since no antigen was added during this second sorting cycle, only cells expressing antibodies that have slow dissociation kinetics remain fluorescent. The plating efficiency of this population was low, presumably due to a combination of potential scFv toxicity (Somerville et al, 1994; Hayhurst and Harris, 1999), Tris-EDTA-lysozyme treatment and exposure to the high shear flow cytometry environment.
  • DNA encoding scFvs was rescued by PCR amplification of the approximately 1 x 10 4 fluorescent events recovered by sorting. It should be noted that the conditions used for PCR amplification result in the quantitative release of cellular DNA from the cells which have partially hydrolyzed cell walls due to the Tris-EDTA-lysozyme treatment during labeling. Following 30 rounds of PCR amplification, the DNA was ligated into pAPExl and transformed into fresh E. coli. A second round of sorting was performed exactly as above, except that in this case only the most fluorescent 2% of the population was collected and then immediately resorted to yield approximately 5,000 fluorescent events.
  • the scFv DNA from the second round was amplified by PCR and ligated into pMoPacl6 (Hayhurst et al, 2003) for expression of the antibody fragments in soluble form in the scAb format.
  • a scAb antibody fragment is comprised of an scFv in which the light chain is fused to a human kappa constant region. This antibody fragment format exhibits better periplasmic solubility compared to scFvs (Maynard et al, 2002; Hayhurst, 2000). 20 clones in the scAb format were picked at random and grown in liquid cultures.
  • periplasmic proteins were isolated and the scAb proteins were rank-ordered with respect to their relative antigen dissociation kinetics, using surface plasmon resonance (SPR) analysis.
  • 11 of the 20 clones exhibited slower antigen dissociation kinetics compared to the 14B7 parental antibody.
  • the 3 scAbs with the slowest antigen dissociation kinetics were produced in large scale and purified by Ni chromatography followed by gel filtration FPLC.
  • all the library-selected clones exhibited excellent expression characteristics and resulted in yields of between 4-8 mg of purified protein per Lin shake flask culture.
  • FIG. 8B a schematic showing the conformation of the 1H, M5, M6 and Ml 8 antibodies is given in FIG. 10.
  • the mutations for M5 were as follows: in the light chain, Q38R, Q55L, S56P, T74A, Q78L and in the heavy chain, K62R.
  • the mutations for M6 were as follows: S22G, L33S, Q55L, S56P, Q78L AND L94 P, and in the heavy chain, S7P, K19R, S30N, T68I and M80L.
  • FIG. 11 shows an alignment of 14B7 scFv (SEQ ID NO:21) and M18 scFv (SEQ ID NO:23) sequences indicating the variable heavy and variable light chains and mutations made.
  • the nucleic acids encoding these sequences are given in SEQ ID NO:20 and SEQ ID NO:22, respectively.
  • the fluorescence intensity of Tris-EDTA-lysozyme permeabilized cells expressing NlpA fusions to the mutant antibodies varied in proportion to the antigen binding affinity.
  • FIG. 8C For example, cells expressing the NlpA- [Ml 8 scFv] protein displayed a mean fluorescence of 250 whereas the cells that expressed the parental 14B7 scFv exhibited a mean fluorescence of 30, compared to a background fluorescence of around 5 (FIG. 8B).
  • Antibodies with intermediate affinities displayed intermediate fluorescence intensities in line with their relative affinity rank. The ability to resolve cells expressing antibodies exhibiting dissociation constants as low as 35 pM provides a reasonable explanation for why three unique very high affinity variants could be isolated and is indicative of the fine resolution that can be obtained with flow cytometric analysis.
  • Ml 8 the highest affinity clone isolated by APEx, contained the S56P mutation but lacked the Q55L substitution found in 1H, M5, and M6.
  • the introduction of this mutation reduced the yield of purified protein more than 5-fold to 1.2 mg/L in shake flask culture.
  • the modified Ml 8 sequence is given in SEQ ID NO:25 and the nucleic acid encoding this sequence is given in SEQ ID NO:24.
  • g3p minor coat protein
  • g3p becomes transiently attached to the inner membrane via its extreme C-terminus, before it can be incorporated onto the growing virion (Boeke and Model, 1982).
  • the antibody fragments are thus both anchored and displayed in the periplasmic compartment. Therefore, the inventors evaluated whether g3p fusion proteins can be exploited for antibody library screening purposes using the APEx format.
  • APEx is based on the anchoring of proteins to the outer side of the inner membrane, followed by disruption of the outer membrane prior to incubation with fluorescently labeled antigen and FC sorting.
  • This strategy offers several advantages over previous bacterial periplasmic and surface display approaches: 1) by utilizing a fatty acylated anchor to retain the protein in the inner membrane, a fusion as short as 6 amino acids is all that was required for the successful display, potentially decreasing deleterious effects that larger fusions may impose; 2) the inner membrane lacks molecules such as LPS or other complex carbohydrates that can sterically interfere with large antigen binding to displayed antibody fragments; 3) the fusion must only traverse one membrane before it is displayed; 4) both N- and C-terminal fusion strategies can be employed; and 5) APEx can be used directly for proteins expressed from popular phage display vectors.
  • APEx can be employed for the detection of antigens ranging from small molecules (e.g. digoxigenin and methamphetamine ⁇ lkDa) to phycoerythrin conjugates (240 kDa).
  • small molecules e.g. digoxigenin and methamphetamine ⁇ lkDa
  • phycoerythrin conjugates 240 kDa
  • the phycoerythrin conjugate employed in FIG. 3B is not meant to define an upper limit for antigen detection, as it is contemplated that larger proteins may be used as well.
  • genes encoding scFvs that bind the fluorescently labeled antigen were rescued from the sorted cells by PCR.
  • An advantage of this approach is that it enables the isolation of clones that are no longer viable due to the combination of potential scFv toxicity, Tris-EDTA-lysozyme disruption, and FC shear forces. In this way, diversity of isolated clones is maximized.
  • Yet another advantage of PCR rescue is that the amplification of DNA from pooled cells can be carried out under mutagenic conditions prior to subcloning.
  • PCR conditions that favor template switching among the protein encoding genes in the pool may be employed during the amplification step to allow recombination among the selected clones. It is likely that PCR rescue would be advantageous in other library screening formats as well.
  • An important issue with any library screening technology is the ability to express isolated clones at a high level.
  • Existing display formats involve fusion to large anchoring sequences which can influence the expression characteristics of the displayed proteins. For this reason, scFvs that display well may not necessarily be amenable to high expression in soluble form as non-fusion proteins (Hayhurst et al, 2003).
  • anthracis protective antigen exhibiting KD values as low as 21 pM.
  • the scFv binding site exhibiting the highest affinity for PA has been humanized, converted to full length IgG and its neutralizing potential to anthrax intoxication is being evaluated in preclinical studies.
  • affinity maturation APEx can be exploited for several other protein engineering applications including the analysis of membrane protein topology, whereby a scFv antibody anchored in a periplasmic loop is able to bind fluorescent antigen and serves as a fluorescent reporter, and also, the selection of enzyme variants with enhanced function.
  • APEx can be readily adapted to enzyme library sorting, as the cell envelope provides sites for retention of enzymatic catalytic products, thereby enabling selection based directly on catalytic turnover (Olsen et al, 2000).
  • the inventors are also evaluating the utilization of APEx for the screening of ligands to membrane proteins.
  • anchored periplasmic expression has the potential to facilitate combinatorial library screening and other protein engineering applications.
  • the leader peptide and first six , amino acids of the mature NlpA protein flanked by Ndel and Sfil sites was amplified by whole cell PCR of XLl-Blue (Stratagene, CA) using primers BRH#08 5'-GAAGGAGATATACATATGAAACTGACAACACATCATCTA-3' (SEQ ID NO:6) and BRH#09 5'-
  • NlpA fragment was used to replace the pelB leader sequence of pMoPacl (Hayhurst et al, 2003) via Ndel and Sfil to generate pAPExl.
  • scFv specific for digoxin Choen et al, 1999
  • Bacillus anthracis protective antigen PA Maynard et al, 2002
  • methamphetamine were inserted downstream of the NlpA fragment in pAPExl via the non-compatible Sfil sites.
  • E. coli ABLE CTM (Stratagene) was the host strain used throughout. E. coli transformed with the pAPExl or pAK200 derivatives were inoculated in terrific broth (TB) supplemented with 2% glucose and chloramphenicol at 30 ⁇ g/ml to an OD600 of 0.1. Cell growth and induction were performed as described previously (Chen et al, 2001). Following induction, the cellular outer membrane was permeabilized as described (Neu and Heppel, 1965).
  • Purified PA protein kindly provided by S. Leppla NTH, was conjugated to BODIPYTM at a 1 to 7 molar ratio with bodipy FL SE D-2184 according to the manufacturers instructions.
  • Unconjugated BODIPYTM was removed by dialysis.
  • Free digoxigenin was removed by dialysis in excess PBS . 4.
  • Affinity Maturation of scFv Libraries with FC Libraries were made from the 14B7 parental scFv using error prone PCR using I standard techniques (Fromant et al, 1995) and cloned into the pAPExl expression vector.
  • CM5 chip Approximately 500RUs of PA was coupled to a CM5 chip using EDC/NHS chemistry. BSA was similarly coupled and used for in line subtraction. Kinetic analysis was performed at 25°C in BIA HBS-EP buffer at a flow rate lOO ⁇ l/min. Five two fold dilutions of each antibody beginning at 20nM were analyzed in triplicate.
  • EXAMPLE 7 Malf Topology Prediction A method was sought for confirming the accuracy of a fluorescent probe-based screening system using E. coli MalF as a model protein, of which intrinsic topology (8 TMHs) has been fully elucidated (Ehrmann et al, 1990).
  • the six representative positions were arbitrarily chosen from MalF polypeptide sequence as follows: Y62, G313 and G407 (the positions located in the known cytoplasmic loops), and V175, M350 and A461 (the positions located in periplasmic loops).
  • a series of expression plasmids were constructed bearing the truncated MalF fused with either of the two topology-reporter candidates at the defined positions.
  • primers 2610-F1 and 2610-R1 were used and pHEN2.dig26-10 containing 26-10 scFv gene (Chen et al, 2001) was used as a template DNA.
  • primers GFP-F1 and GFP-R1 were used for the amplification of GFP.
  • pGFPmut2 containing a GFP variant gene was used as a template DNA.
  • Each PCR product was digested by two restriction enzymes (BglE and EcoRI).
  • FIG. 12 shows the simple structure of MalF-fused expression system and fusion points in MalF protein.
  • GFP-F1 CGAAGCTTAGATCTAGTAAAGGAGAAGAACTTT (SEQ ID NO:35)
  • GFP-R1 GGAATTCTTTGTATAGTTCATCCATGCC (SEQ ID NO:36)
  • IPTG isopropyl ⁇ -D-thiogalactoside
  • 0.1 mL of cells from the flask culture were mixed with 100 nM of digoxin-BODIPY probe in 0.9 mL of 5X PBS (phosphate buffered saline) and after 1 hr of incubation at room temperature with shaking, the cells were collected by centrifugation. The cells were resuspended in 1 mL of IX PBS and a 5 ⁇ L aliquot was diluted into 2 mL of IX PBS and labeled with propidium iodine (PI) for flow cytometric detection of nonviable cells.
  • 5X PBS phosphate buffered saline
  • the clones with the cytoplasmic reporter (GFP) fused at MalF cytoplasmic loops showed nearly 20 fold higher fluorescence-mean values than those fused at the periplasmic loops (V175, M350, or A461).
  • the 26-10 scFv conferred distinguishable fluorescent signals to the host cells only when it was fused at either of the known periplasmic loops.
  • the clones with the periplasmic reporter fused at the cytoplasmic loop had a minor population that shows a higher fluorescent intensity, such clones with noise fraction could be eliminated by the repetitive sorting of the selected clones.
  • TatC Topology prediction TatC (259 amino acids in length) is a conserved inner membrane protein (IMP) that plays a pivotal role in the TAT (Twin- Arginine Translocation) system (Berks, 1996). In E. coli, four integral membrane proteins, TatA, TatB, TatC, and TatE are involved in the system.
  • IMP conserved inner membrane protein
  • TatA and TatB share a similar and simple overall structure with single-span TMH and an amphipathic ⁇ -helix at their C- terminal region
  • TatC is predicted to be a polytopic membrane protein with 6 TMHs, of which N- and C-termini are exposed to the cytoplasmic face (Buchanan et al, 2002; Drew et al, 2001).
  • An alternative, conflicting model for TatC topology has been reported by
  • plasmid pTOPO-2610 was constructed by cloning the 26-10 scFv gene (Chen et al, 2001) using the primers Topo-F (5'- GCACTAGTAGATCTCATATGGAGCCCGGGCATCCGGGGAGCTC -3' (SEQ ID NO:37)) 5 Topo-2610-F (5'- CGGGCATCCGGGGAGCTCAGGCCCAGCCGGCCATG -3' (SEQ ID NO:38)) and Topo-2610-R (5'- GGCGAATTCGGCCCCCGAGG -3' (SEQ ID NO:39)), which introduced the unique restriction sites of Spel and EcoRI (underlined) at the 5' and 3 '-ends of the 26-10 scFv gene, respectively, and enabled cloning of this sequence into Xbal-EcoRI digested pMoPacl.
  • Plasmid pTOPO-GFP was constructed by cloning the GFPmut2 variant (Cormack et al, 1996) using the primers Topo-F, Topo-GFP-F (5'- CGGGCATCCGGGGAGCTCCAATGAGTAAAGGAGAAGAACTTT -3 '(SEQ ID NO:40)) and Topo-GFP-R (5'- GCGAATTCTTTGTATAGTTCATCCATGCC-3'(SEQ ID NO:41)). The Spel-EcoRI digested PCR product was then cloned into Xbal-EcoRI digested pMoPacl.
  • tatC fused with reporter gene was carried out based on the method termed THIO-ITCHY (Lutz et al, 2001) with some modifications.
  • the entire sequence for the tatC coding region was amplified using gene- specific primers TatC-F (5'- GGCGGTACCGAAGATCTGAAGGAGATATACACATGTCTGTAGAAGATACTC • - 3 '(SEQ ID NO:42)) and TatC-R (5'- CCTGACGGGCGGTTGAATTTCTTCTTCAGTTTTTTCGCTTTCT -3 '(SEQ ID NO:43)), where the underlined sequences indicate Kp ⁇ l and BglH restriction sites, with the existence of appropriate concentration (20 ⁇ M) of ⁇ -phosphothioate dNTPs.
  • the amplified thio-tatC fragments were subjected to treatment with exonuclease III, followed by mungbean nuclease trimming to give blunt-ended DNA fragments with various C-terminal endpoints.
  • the blunt-ended fragments were digested by Bgl ⁇ l, then ligated with the Bgl ⁇ l- Smal fragment of pTOPO-GFP or that of pTOPO-26.10, followed by transformation of E. coli Judel (DH10B derivative that harbors the F' factor derived from XLl-blue) to make the fusion libraries (2.0xl0 4 and 3.0xl0 4 independent colonies for 26-10 scFv and GFP fusion libraries, respectively).
  • FIG. 14 shows the simple diagram of library construction. The plasmids were then purified from the E. coli Judel library and transformed into E. coli MC4100, which was used as a host cell for screening the library. Cell cultures and sample preparations were accomplished using the methods described in the previous example.
  • Sorting by flow cytometry was performed with a Becton-Dickinson FACS caliber, and the desired cell population was gated by setting appropriate SSC (side-scattered light), FSC (Forward-scattered light), FL1, and FL2 windows (FL1 is used to monitor GFP fluorescence and FL2 is used to monitor PI fluorescence) and cells were sorted in exclusion mode.
  • SSC side-scattered light
  • FSC Forward-scattered light
  • FL1 Forward-scattered light
  • FL1 FL1 is used to monitor GFP fluorescence
  • FL2 is used to monitor PI fluorescence
  • the cells grown on the plate were collected and inoculated into new TB medium, and then sorted using the methods described above. After the second round of sorting and incubation on agar plates, forty-eight clones out of the resulting ca. 3,000 colonies were randomly chosen. The 48 clones were then subjected to the analysis of monoclonal fluorescent intensity using the same induction and sorting conditions that were used in the selection rounds. Every clone among the selected 48 clones showed significantly higher (from 15 to 100' times) fluorescent mean-values than that of the negative control (host cell without plasmid). Sequences of the sorted cells were determined by using API Prism 3700 (Applied Biosystems, Foster city, CA).
  • DNA sequencing analysis of the 48 clones allowed identification of 31 distinctive fusion points on the primary amino-acid sequence of TatC.
  • the identified fusion points were present in (i) N- and C- terminal regions, (ii) a stretch between the putative 2nd and 3rd TMHs, or (iii) another stretch between the putative 4th and 5th TMHs without any exceptions.
  • the deduced topology model of TatC was delineated as shown in FIG. 15. The information for the positions and frequencies of the identified fusion points are also embedded in FIG. 15. The deduced structure was perfectly consistent with the originally predicted 6 TMHs model, rather than the 4 TMHs model (Gouffi et al, 2002).
  • the cultured cells expressing TatC fused 26-10 scFv were labeled with the digoxin-BODIPY probe (100 nM) in IX PBS buffer. After labeling for 45 min at room temperature, sorting by flow cytometry was performed with a Becton-Dickinson FACS caliber, and the desired cell population was gated by setting appropriate SSC, FSC, FL1, and FL2 windows (FL1 is used to monitor digoxin-BODIPY fluorescence and FL2 is used to monitor PI fluorescence) and cells were sorted in exclusion mode. After the second round of sorting and incubation on agar plates containing 35 ⁇ g/mL of chloramphenicol at 30°C, forty clones out of the resulting ca.
  • both fusion clones showed the clear differences in their fluorescent intensities, which were well matched to the 6 TMH topology model.
  • GFP fusion clones at El 87 and LI 89 positions exhibited higher fluoresecent intensities than the other clones.
  • 26-10 scFv fusion clones at the same fusion positions showed much lower fluorescent intensities even though their intensities were still higher than that of negative control (A98).
  • A98 negative control
  • GFP fusion showed some intermediate intensities and 26-10 scFv fusions showed lower signal similar to negative control (A98). This data indicated that the A200 position is placed inside the membrane, which is in agreement with the 6 TMHs model.
  • NlpA is a lipoprotein that localizes to the inner membrane. Since the first amino acid (Cys) of NlpA is necessary for lipidation with lipid of the inner membrane, only the next five amino acids (DQSSS) were randomized. In this library, some protein may be localized in the inner membrane, some protein may be localized in the outer membrane, and some protein may be present in the periplasm without localizing to either membrane.
  • Plasmid p26-10 scFv APEx which contains the native NlpA sequence (CDQSSS), was used as template DNA for the PCR reaction using the three primers named above.
  • the PCR products were digested with the Fold restriction enzyme, which can create the EcoRI-cut cohesive end (AATT, underlined above) and Notl-cut cohesive end (GGCC, underlined above), and the digested D ⁇ A was cloned into p26-10 scFv AP ⁇ x digested with EcoRI and Notl restriction enzymes.
  • the simple structure of the library vector is shown in FIG. 17. From the library (in E. coli Judel strain), 16 clones were randomly chosen and their sequences for 5 amino acids were determined. All 16 clones showed fully randomized sequences in the 5 amino acids after the first Cys residue. This library was then used to screen for sequences useful for tethering proteins onto the inner membrane.
  • EXAMPLE 10 Screening of Sequences for Tethering Proteins on the Membranes by Flow Cytometry. Overnight cultures of the E. coli Judel library were subcultured into fresh TB medium at 37°C. After 2 hr, the flask was moved to a 25°C shaking water bath to decrease the culture temperature. After 30 min cooling at 25 °C, induction was done with 1 mM IPTG. After 4hr, cells were collected and spheroplasts were prepared by lysozyme-EDTA treatment to remove the unbound 26-10 scFv from the periplasm as well as the outer membrane bound 26-10 scFv.
  • the collected cells were resuspended in a buffer (350 ⁇ L) containing 0.1 M Tris-Cl (pH 8.0) and 0.75 M sucrose, and then 700 ⁇ L of ImM NaEDTA was added. Lysozyme (Sigma) was added to 100 ⁇ g/mL and cells were incubated at 37°C for 10 min. Finally, 50 ⁇ L of 0.5 M MgCl was added and further incubated on ice for 10 min. The spheroplast cells were then pelleted by 10 min of centrifugation at 10,000 rpm and then resuspended in IX PBS buffer (phosphate buffered saline).
  • IX PBS buffer phosphate buffered saline
  • spheroplast cells were mixed with 100 nM of Bodipy-Digoxigenin conjugate probe (Molecular Probes, USA) in 0.9 mL of IX PBS and after 30 min of incubation at room temperature with shaking, the cells were collected by centrifugation. The cells were resuspended in 1 mL of IX PBS and a 5 ⁇ L aliquot was diluted into 2 mL of IX PBS buffer prior to sorting using a Moflo flow cytometry (Darko Cytomation, USA).
  • the desired cell population was gated by setting appropriate SSC (side- scattered light), FSC (Forward-scattered light), and FL1 windows (for Bodipy fluorescence) and cells were sorted in purify 1 and 2 drop mode.
  • a total of 9 x 10 7 bacteria were sorted using an ultra-high throughput Cytomation hie.
  • MoFlo droplet deflection flow cytometer selectively gated for high BODIPYTM fluorescence.
  • Approximately 2% of the cells sorted with the highest 530nm fluorescence (FL1) were collected and immediately resorted as above.
  • DNA encoding scFvs was rescued by PCR amplification of the approximately 7 x 10 4 fluorescent events recovered by sorting. In this PCR reaction, two primers, NlpARan-Fok-Fl
  • GGACTGATGGATGTACGAATTTCTAGAGAAGGAG SEQ ID NO:48
  • NLPA- RAN-R2 GGACTGATGGATGGATTTGATCJCGAGCTTGG
  • the PCR product was digested with Fokl restriction enzyme, which can create the Xbdl-c t cohesive end (CTAG, underlined above) and Xhol-cw ⁇ cohesive end (TCGA, underlined above), and the digested DNA was cloned into p26-10 scFv APEx digested with the Xbal and Xh ⁇ l restriction enzymes.
  • a second round of sorting was then performed as described above, followed by the PCR reaction and cloning, also as described above.
  • 100 clones were then randomly chosen for the analysis of their fluorescence by Flow cytometry. As shown in Table 3, among 100 clones, 12 clones showed higher or similar fluorescence as compared to the native NlpA sequence (CDQSSS). The high fluorescence indicates the anchoring of 26-10 scFv to inner membrane. This data thus shows that the APEx system can be used to discover sequences useful for tethering proteins onto the inner membrane.
  • Table 3 The amino acid sequences in randomized region and relative fluorescence of sorted clones.
  • Boder et al Proc. Natl. Acad. Sci. USA, 97:10701-10705, 2000. Bodine and Ley, EMBO J, 6:2997, 1987.

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Abstract

La présente invention permet de surmonter les lacunes de l'art antérieur en proposant une approche rapide pour l'isolation de polypeptides capables d'ancrage de polypeptides hétérologues à une membrane interne bactérienne. Dans cette technique, des bibliothèques de polypeptides d'ancrage candidats sont exprimés sous forme de fusions avec un polypeptide hétérologue qui est capable d'être détecté lors de sa liaison à une membrane interne. Dans des bactéries exprimant une séquence d'ancrage fonctionnelle, le polypeptide hétérologue se lie à la face extérieure de la membrane interne. Des bactéries avec la séquence d'ancrage fonctionnelle peuvent être identifiées par l'élimination de la membrane externe pour l'élimination du polypeptide hétérologue non ancré suivi de la détection de polypeptide hétérologue ancré. De telles bactéries peuvent être détectées de plusieurs manières, y compris l'utilisation de la fluorescence directe ou d'anticorps secondaires qui sont marqués par fluorescence, permettant ainsi l'utilisation de techniques efficaces telles que le tri cellulaire activé par la fluorescence.
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