EP1438584A4 - Target-assistiertes iteratives screening (tais): ein neues screening-format für grosse molekulare repertoires - Google Patents

Target-assistiertes iteratives screening (tais): ein neues screening-format für grosse molekulare repertoires

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Publication number
EP1438584A4
EP1438584A4 EP02783996A EP02783996A EP1438584A4 EP 1438584 A4 EP1438584 A4 EP 1438584A4 EP 02783996 A EP02783996 A EP 02783996A EP 02783996 A EP02783996 A EP 02783996A EP 1438584 A4 EP1438584 A4 EP 1438584A4
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EP
European Patent Office
Prior art keywords
target
proteins
protein
members
library
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Withdrawn
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EP02783996A
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English (en)
French (fr)
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EP1438584A1 (de
Inventor
Alexei Kourakine
Dale Bredesen
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Buck Institute for Research on Aging
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Buck Institute for Research on Aging
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Publication of EP1438584A1 publication Critical patent/EP1438584A1/de
Publication of EP1438584A4 publication Critical patent/EP1438584A4/de
Withdrawn legal-status Critical Current

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Classifications

    • C—CHEMISTRY; METALLURGY
    • C40—COMBINATORIAL TECHNOLOGY
    • C40B—COMBINATORIAL CHEMISTRY; LIBRARIES, e.g. CHEMICAL LIBRARIES
    • C40B30/00—Methods of screening libraries
    • C40B30/04—Methods of screening libraries by measuring the ability to specifically bind a target molecule, e.g. antibody-antigen binding, receptor-ligand binding
    • 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
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6883—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
    • 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
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G01N33/6803—General methods of protein analysis not limited to specific proteins or families of proteins
    • G01N33/6845—Methods of identifying protein-protein interactions in protein mixtures

Definitions

  • TAIS TARGET ASSISTED ITERATIVE SCREENING
  • This invention pertains to the field of proteomics.
  • this invention pertains to a dual screening method for determining interactions between members of a library and various targets that allows simultaneous screening for large numbers of interactions (e.g. protein-protein interactions) between library members and the target(s).
  • the present invention pertains to a novel, rapid in vitro screening method for the identification and characterization of protein-protein interactions (e.g. interactions mediated by specialized protein modules such as SH3, PDZ and WW domains).
  • the method is well suited to large-scale functional genomics approaches. In essence the present method combines the advantages of phage display technology and cDNA expression libraries.
  • this invention provides a method of identifying interacting proteins from a plurality of potentially interacting proteins. The method typically involves i) contacting one or more targets (e.g.
  • target proteins with a protein display library comprising a plurality of potential binding proteins for the one or more target proteins; ii) selecting members of the protein display library that bind to the one or more target proteins to provide a preselected set of potential binding proteins; iii) separating the members of the preselected set of potential binding proteins from the bound target protein and localizing and/or immobilizing the members on a solid support such that the members are spatially addressable; and iv) contacting members of the preselected set of potential binding proteins with one or more target proteins; and v) detecting binding of members of the preselected set of potential binding proteins with the one or more target proteins whereby binding of a member of said set of potential binding partners with a target protein indicates that the member and the target protein are interacting proteins.
  • the target proteins are attached to a solid support during the first contacting step.
  • the protein display library can be any convenient display library.
  • Preferred display libraries include, but are not limited to phage display, bacterial display, yeast display, eukaryotic virus display library, direct plasmid display library, and so forth.
  • the library is an in vitro display library (e.g. covalent display technology (CDT), polysome display, eukaryotic in vitro transcription/translation systems, RNA-peptide fusions, and the like).
  • Such libraries typically comprise at least 100 different members, preferably at least 1000 different members, more preferably at least 10,000 and most preferably at least 10 6 , 10 7 , 10 s , 10 9 or 10 10 different members.
  • the library displays a cDNA library (e.g. from a particular organism, tissue, cell type, etc.).
  • amplification of preselected subset of potential interactors of the target(s) is often performed, and can be performed in a spatially addressable manner.
  • the "separating" comprises amplifying members of the protein display library that bind to said one or more target proteins and/or the separating and/or immobilizing comprises amplifying members of the protein display library that bind to said one or more target proteins.
  • the amplifying can comprise amplification of the members when they are spatially separated and addressable.
  • the selecting comprises removing unbound members of the display library from the solid support.
  • the selecting can comprise capturing one or more target proteins and/or bound library members (i.e. in a bound complex) using an affinity matrix.
  • contacting members of the preselected set of potential binding partners with one or more target proteins comprises adsorbing members of the preselected set of potential binding partners to a solid support (e.g. a membrane).
  • the detecting can be by means of a label attached to the target protein(s).
  • Preferred labels include, but are not limited to a fluorescent label, a radioactive label, an enzymatic label, a colorimetric label, and a magnetic label.
  • the contacting of step (i) comprises contacting the one or more target proteins with a protein display library where said one or more target proteins are attached to a solid support; the contacting of step (iv) comprises attaching members of the preselected set of potential binding proteins to a solid support to provide a set of attached preselected potential binding proteins and contacting the attached preselected potential binding proteins with the one or more target(s) (e.g. target proteins).
  • the target proteins used in the contacting of step (iv) can be labeled with a detectable label before, during, or after the target proteins are contacted to the preselected potential binding proteins.
  • the method further comprises sequencing the nucleic acid encoding the displayed protein on a member of the preselected display library that binds to the target protein.
  • the contacting of step (i) comprises contacting one or more target proteins with a protein display library where said one or more target proteins and the protein display library are in solution.
  • the selecting step can comprise capturing target proteins bound to members of the protein display library using an affinity matrix that specifically binds the target proteins or a tag attached to the target proteins.
  • the contacting of step (iv) can comprise attaching members of said preselected set of potential binding proteins to a solid support to provide a set of attached preselected potential binding proteins and contacting the attached preselected potential binding proteins with the one or more target proteins.
  • the detecting comprises determining the amino acid sequence of a member of the set of potential binding partners (e.g., binding proteins) that binds a target protein.
  • the method can further involve recording the amino acid sequence or identity of a member of the set of potential binding partners that binds a target protein in a database of proteins that interact with the target.
  • any target moiety can be used.
  • Such moieties include, but are not limited to various natural or synthetic chemical compounds including, but not limited to drugs, small organic molecules, nucleic acids, proteins, glycoproteins, carbohydrates, and the like.
  • the display library need not be limited to proteins. Virtually any moiety that can be displayed in a library is suitable.
  • Particularly preferred display libraries include, but are not limited to protein or nucleic acid display libraries.
  • the method typically comprises, i) contacting one or more target moieties with the library; ii) selecting members of the library that bind to the one or more target moieties to provide a preselected set of potential binding partners; iii) separating the members of the preselected set of potential binding partners from the bound target and immobilizing the members on a solid support such that the members are spatially addressable; iv) contacting members of the preselected set of potential binding partners with one or more target moieties; and v) detecting binding of members of the set of potential binding partners with said one or more target moieties whereby binding of a member of the set of potential binding partners with a target binding moiety indicates that said member is a binding partner that interacts with the target moiety.
  • Preferred libraries include, but are not limited to a phage display library, a bacterial display library, a yeast display library, a eukaryotic virus library, a direct encoded plasmid library, and the like.
  • the library is an in vitro display library (e.g. a covalent display technology (CDT) library, a polysome display library, an RNA-peptide fusion library, etc.).
  • the target moiety is a nucleic acid (e.g. a DNA, an RNA), a lipid, a carbohydrate, a glycoprotein, or a small organic molecule.
  • kits practicing any of the methods described herein.
  • the kit comprises a protein display library; and instructional materials providing protocols for the methods described herein.
  • TAIS eliminates the loss of weaker binders and propagation biases, that result from competition between individual phage during repetitive selection-amplification cycles.
  • the method permits screening of significantly larger libraries than the ones routinely used in cDNA expression library screening.
  • the upper limit on the size of the library used in TAIS is defined by existing technologies of phage display library preparation, i.e., on the order of 10 8 -10 12 or more phage.
  • TAIS provides a number of advantages: The method does not require costly and sophisticated equipment, and can be used with commercially available reagents. The method involves only simple biochemical and microbiological manipulations, and, additionally because of the low cost is easily attainable for almost any lab, with minimal investment for setup. The method has a short turnaround time: normally within 24 hours an investigator will know whether or not a particular screen has been successful, and often, in 48 to 72 hours an investigator has DNA ready for sequencing to analyze the cDNAs selected in the screen. The screening is performed in vitro, i.e., under defined and manipulatable conditions; the readout is direct, and is easily accurately quantitated. The method provides a powerful tool to characterize ligand preferences of peptide recognition domains.
  • cDNA libraries e.g. phage-displayed cDNA libraries
  • the lengths of the peptides in the library are not fixed.
  • the libraries can feature natural peptide ligands of the target that provide internal references for physiologically relevant affinities and specificities of the interaction in question.
  • TAIS allows the analysis of relatively weak and/or poorly propagating binders that are typically lost during the standard phage display panning procedure. Propagation biases and disparity in stabilities between different phages are of
  • the screening method can be applied to a number of other large molecular diversities such as phage-displayed peptide and recombinant antibody libraries, cell displayed polypeptide libraries, etc. Iterative presentation of the target in two different molecular contexts facilitates minimization of non-specific interactions.
  • the methods of this invention involve two screening steps.
  • the methods comprise: i) contacting one or more target proteins with a molecular library (e.g. a protein display library, nucleic acid display library) comprising a plurality of potential binding partners for the one or more targets (e.g.
  • target proteins ii) selecting members of the display library that bind to the one or more targets to provide a preselected set of potential binding partners; iii) separating said members of said preselected set of potential binding partners from the bound target and immobilizing said members on a solid support such that said members are spatially addressable; and iv) contacting members of the preselected (and optionally amplified) set of potential binding partners with one or more targets again; and v) detecting binding of members of the set of potential binding proteins with the one or more targets whereby binding of a member of the set of potential binding partners with a target indicates that the member and the target interact.
  • the methods of this invention typically involve an initial screen that entails contacting one or more target moieties with a library of potential binding partners (e.g. preferably nucleic acids or proteins).
  • the library is preferably a display library, more preferably a protein display library (e.g. phage display, bacterial display, yeast display, eukaryotic virus display library, direct plasmid display library, etc.).
  • the target moieties can include any moiety that is expect to be bound or is capable of being bound by a protein. Such moieties include, but are not limited to proteins, nucleic acids, lipids, glycoproteins, carbohydrates, polysaccharides, and the like. The target moieties need not be limited to individual molecules. Thus, for example, it is possible to use cell surfaces, receptors, tissues, and the like as targets.
  • the target moieties are typically contacted with a library of potential binding partners (e.g. proteins that might be capable of binding to the target(s)).
  • a library of potential binding partners e.g. proteins that might be capable of binding to the target(s)
  • Such libraries typically comprise at least 100 different members, preferably at least 1000 different members, more preferably at least 10,000 and most preferably at least 10 6 , 10 7 , 10 8 , 10 9 or 10 10 different members.
  • the libraries are cDNA libraries derived from a particular cell type/line, and/or a particular tissue, and/or a particular organism. The libraries, however, need not be limited to cDNA libraries.
  • Other libraries include, but are not limited to antibody libraries (e.g. single chain antibody libraries), libraries of proteins randomized in one or more domains, libraries comprising shuffled polypeptides, and the like.
  • the libraries of potential binding partners are provided on a "display vector".
  • display vectors include, but are not limited to phage- display vectors, bacterial display vectors (Fuchs et al. (1991) Biotechnology 9, 1369-1372), yeast display libraries (Boder and Wittrup (1997) Nat. Biotechnol. 15: 553-557), eukaryotic virus libraries (Kasahara et al. (1994) Science 266: 1373-1376), and direct plasmid display libraries (Cull et al. (1992) Proc. Natl. Acad. Sci. U. S. A. 89: 1865-1869), and the like.
  • Suitable libraries also include in vitro display technologies (e.g.
  • CDT covalent display technology
  • polysome display eukaryotic in vitro transcription/translation systems
  • R ⁇ A- peptide fusions e.g., R ⁇ A- peptide fusions, and the like (see, e.g., Fitzgerald (2000) Drug Discovery Today 5(6): 253- 258, and references cited therein).
  • polypeptides on the surface of bacteria or of viruses that infect bacteria makes it possible to screen and one or more binding polypeptide or a libraries of greater than 10 10 clones.
  • phage display a nucleic acid encoding the polypeptide is inserted into the gene encoding a phage surface protein (e.g., pill) and the polypeptide-surface fusion protein is displayed on the phage surface (McCafferty et al. (1990) Nature, 348: 552-554; Hoogenboom et al. (1991) Nucleic Acids Res. 19: 4133-4137).
  • phage bearing binding polypeptides can be separated from non-binding phage by binding to a target (e.g. via antigen affinity chromatography) (see, e.g., McCafferty et al. (1990) Nature, 348: 552-554).
  • a target e.g. via antigen affinity chromatography
  • Phage display has been successfully applied to a wide range of peptides and proteins, including antibodies McCafferty et al. (1990) Nature, 348: 552-554), growth hormone (Bass et al. (1990) Proteins: Struct. Funct. Genet. 8(4): 309-314), DNA binding proteins (Jamieson et al. (1994) Biochem., 33(19): 5689-5695), enzymes (McCaffety et al. (1991) Protein Eng., 4(8): 955-961); Corey et al. (1993) Gene, 128(1): 129-134);
  • a phage display library utilizes so called
  • hyperphage In hyperphage, the number of single-chain antibody fragments (scFv) or other proteins, presented on filamentous phage particles can be increased by more than two orders of magnitude by using a newly developed helper phage (hyperphage).
  • hyperphage have a wild-type pIII phenotype and are therefore able to infect F+ Escherichia coli cells with high efficiency; however, their lack of a functional pill gene means that the phagemid- encoded pffl-antibody fusion is the sole source of pHI in phage assembly. This results in a considerable increase in the fraction of phage particles carrying an the inserted protein on their surface (see, e.g., Rondot et al. (2001) Nature Biotechnology, 19(1): 75-78). [0055] Similar to phage-display systems, methods are known to display heterologous proteins on the surface of bacteria. Thus, for example, U.S. patent 6,190,662
  • bacterial systems comprise tripartite chimeric genes.
  • One segment of the tripartite gene is a targeting DNA sequence encoding a polypeptide capable of targeting and anchoring the fusion polypeptide to a host cell outer membrane. Targeting sequences are well known and have been identified in several of membrane proteins including Lpp.
  • the Lpp targeting sequence includes the signal sequence and the first 9 amino acids of the mature protein. These amino acids are found at the amino terminus of Lpp.
  • E. coli outer membrane lipoproteins from which targeting sequences may be derived include TraT, OsmB, NlpB and BlaZ. Lipoprotein 1 from Pseudomonas aeruginosa or the PAl and PCN proteins from Haemophilus influenza as well as the 17 kDa lipoprotein from Rickettsia rickettsii and the H.8 protein from Neisseria gonorrhea and the like can be used.
  • a second component of the tripartite chimeric gene is a DNA segment encoding a membrane-trans versing amino acid sequence.
  • Transversing is intended to denote an amino acid sequence capable of transporting a heterologous or homologous polypeptide through the outer membrane.
  • the membrane transversing sequence will direct the fusion polypeptide to the external surface.
  • transmembrane segments are typically found in outer membrane proteins of all species of gram-negative bacteria. Transmembrane proteins, however, serve a different function from that of targeting sequences and generally include amino acids sequences longer than the polypeptide sequences effective in targeting proteins to the bacterial outer membrane. For example, amino acids 46-159 of the E.
  • coli outer membrane protein OmpA effectively localize a fused polypeptide to the external surface of the outer membrane when also fused to a membrane targeting sequence.
  • These surface exposed polypeptides are not limited to relatively short amino acid sequences as when they are incorporated into the loop regions of a complete transmembrane lipoprotein.
  • the third gene segment comprising the tripartite chimeric gene fusion is a
  • DNA segment that encodes any one of a variety of desired heterologous polypeptides is provided.
  • Suitable display systems include, but are not limited to various in vitro display technologies such as covalent display technology (CDT), polysome display, eukaryotic in vitro transcription/translation systems, RNA-peptide fusions, and the like (see, e.g., Fitzgerald (2000) Drug Discovery Today 5(6): 253-258, and references cited therein).
  • CDT covalent display technology
  • polysome display eukaryotic in vitro transcription/translation systems
  • RNA-peptide fusions e.g., RNA-peptide fusions, and the like.
  • CDT exploits the properties of a replication initiator protein from the E. coli bacteriophage P2.
  • the protein is the product of the viral Agene (P2A) and is an endonuclease that initiates a rolling circle replication process by binding to the viral origin (on) and introducing a single strand discontinuity (nick) in the DNA.
  • P2A viral Agene
  • the 3'-OH group that is exposed by the action of P2A is used to prime progeny DNA synthesis using the host replication machinery (Schnos and Inman (1971) /. Mol. Biol. 55: 31-38; Geisselsoder (1976) J. Mol. Biol. 100: 13-22; Chattoraj (1978) Proc. Natl. Acad.
  • the nicking event also exposes a 5' phosphate and this becomes covalently attached to a tyrosine residue in the active site of P2A (Lindahl (1970) Virology 42: 522-533; Liu et al. (1994) Nucleic Acids Res. 22: 5204-5210).
  • P2A exclusively attaches to the same molecule of DNA from which it has been expressed.
  • the high fidelity of the cis activity and the fact that the recognition sequence for the covalent attachment, on, occurs within P2A's own coding sequence Schott al. (1994) Nucleic Acids Res.
  • a pool of DNA molecules is prepared, each containing the coding sequence of P2A fused to the coding sequence for one of a diverse population of potential binding moieties (linear peptides or protein domains).
  • the DNA pool is transcribed and translated concurrently in vitro using an E. coli S30 lysate and, because of the czsactivity of P2A, each DNA molecule becomes covalently tagged with its own
  • the protein-DNA complexes are then subjected to various screening/ selection strategies.
  • Polysome display systems work by transcribing and translating DNA templates in vitro under conditions that enable the isolation of stable mRNA-ribosome- nascent polypeptide complexes (Schaffitzel et al. (1999) J. Immunol. Methods 231: 119- 135). This is achieved by controlling the concentration of magnesium ions (to stabilize the ribosome particle) and by either terminating polypeptide elongation by the addition of chloramphenicol or cooling down the translation products of mRNA templates that lack stop codons .
  • Target-specific polysome complexes are retained on an appropriately derivatized solid surface and the co-selected mRNAs released by dissociation of ribosomes using ethylene diamine tetraacetate (EDTA). These are then recovered by reverse transcription (RT) and PCR for further manipulation.
  • EDTA ethylene diamine tetraacetate
  • Another in vitro display system uses a puromycin molecule to provide a covalent linkage between mRNA molecules and their encoded polypeptides (Roberts and Szostak (1997) Proc. Natl. Acad. Sci., USA, 94: 12297-12302).
  • Puromycin is an antibiotic that mimics the aminoacyl end of tRNA and functions by entering the ribosomal A-site and forming an amide linkage with nascent polypeptide through the peptidyl transferase activity of the ribosome.
  • the puromycin is attached to the 3' end of a single-stranded DNA linker that is in turn ligated to the 3' end of the library-encoding mRNA.
  • a ribosome reaches the junction between the mRNA and the DNA linker and stalls.
  • the puromycin can then enter the ribosomal A-site and form a stable amide linkage with the encoded peptide.
  • a library pool of mRNA-DNA- puromycin molecules can therefore be translated in vitro and purified RNA-peptide complexes incubated with a target molecule for screening. As with the polysome display system, retained complexes are recovered for further manipulation by RT-PCR.
  • display libraries are created that express a library of cDNAs, or other potential binding proteins as described herein.
  • cDNAs encoding all the desired potential binding proteins can be prepared and inserted into the "vehicle(s) comprising the display library.
  • the inserted nucleic acids are made according to methods well known to those of skill in the art.
  • the nucleic acids can be chemically synthesized using nucleotide reagents.
  • the nucleic acids are created using standard cloning techniques, e.g., amplification (e.g., PCR) cloning with appropriate primers.
  • amplification e.g., PCR
  • Selecting bound members of the phage- or bacterial-display library are selected to provide a preselected set of potential binding proteins. Methods of selecting bound phage-display or bacterial display members or other display library members are well known to those of skill in the art.
  • the target moiety e.g. protein, DNA, etc.
  • the target moiety is provided attached to a solid support/substrate.
  • the unbound phage can be washed away and/or the substrate bearing the target(s) bound by phage can be separated from the solution containing the library. Repetitive wash steps will eliminate unbound library members.
  • Suitable supports for the attachment of target moieties include, but are not limited to the surfaces of wells, capillaries, planar surfaces, particulate materials (beads, etc), slurries, gels, and the like.
  • Preferred materials include, but are not limited to magnetic beads, glass, plastic, ceramics, metals, various resins, membranes, and the like.
  • the target moiety is coupled to the surface according to standard methods well known to those of skill in the art.
  • the target moieties can be directly coupled to the substrate or can be joined to the substrate through a linker.
  • the procedure for attaching a target moiety to the substrate will vary according to the chemical structure of the moiety.
  • Proteins contain a variety of functional groups (e.g., -OH, -COOH, -SH, or -NH 2 ) groups, that are available for reaction with a suitable functional group on a surface or a linker to bind the target thereto.
  • the target moiety can be derivatized to expose or attach additional reactive functional groups.
  • the derivatization may involve attachment of any of a number of linker molecules such as those available from Pierce Chemical Company, Rockford Illinois.
  • a bifunctional linker having one functional group reactive with a group on a particular target moiety and another group reactive with a group on the substrate can be used to anchor the target moiety.
  • the target moieties can be attached to the surface by simple adsorption.
  • the target moieties can be provided in solution and contacted to the members of the phage- or bacterial display library also in solution.
  • the target moiety can comprise a domain (tag) that can be specifically captured/bound by an affinity reagent (e.g. an antibody, ligand, etc.).
  • an affinity reagent e.g. an antibody, ligand, etc.
  • the target moiety can be attached to a tag (e.g. an affinity tag) that can be captured by an affinity reagent.
  • Affinity tags are well known to those of skill in the art.
  • Such tags include, but are not limited to biotin with avidin/streptavidin, ligands and their cognate receptors, particularly haptens and antibodies, polyhistidine with Ni-NTA, glutathione S-transferase (GST) and glutathione, epitopes and cognate antibodies, and the like.
  • Certain affinity tags include epitope tags.
  • Epitope tags are well known to those of skill in the art.
  • antibodies (intact and single chain) specific to a wide variety of epitope tags are commercially available. These include but are not limited to antibodies against the DYKDDDDK (SEQ ID NO: 5) epitope, c-myc antibodies (available from Sigma, St. Louis), the HNK-1 carbohydrate epitope, the HA epitope, the HSV epitope, the His 4 , His 5 , and His 6 epitopes that are recognized by the His epitope specific antibodies (see, e.g., Qiagen), and the like.
  • the target moiety is tagged with a hexahistidine (His 6 ) epitope tag that is bound by a Cu, Ni, or Co complex.
  • His 6 hexahistidine
  • One particularly preferred complex for binding His 6 tags is Ni-NTA (Ni- nitrilotriacetic acid).
  • the affinity tag is a biotin which can then be captured by avidin, streptavidin, or variants thereof.
  • the affinity tagged target moiety is contacted with the phage- or bacterial display library, e.g., in solution. Where suitable binding polypeptides exist in the library the target moieties are bound thereby forming a target moiety/binding polypeptide complex.
  • the bound complexes can be recovered from solution phase by the use of an affinity matrix (e.g. a resin or other substrate attached to a ligand that binds to the affinity tag on the target moieties). Once isolated, the assay proceeds as with the target moieties provided attached to a substrate.
  • the target moieties binding polypeptides are isolated thereby providing a preselected set of potential binding proteins.
  • the bound library members can then be separated (e.g. eluted) from the target moieties by the use of standard methods well known to those of skill in the art (e.g. using denaturing reagents, high salt, chaotropic reagents, and the like).
  • the methods of this invention involve a second screening assay.
  • the preselected set of potential binding partners is again probed with the one or more target moieties to identify which members of the potential binding partners bind (e.g. specifically bind) to particular target moieties.
  • the second assay is a different format from the first assay.
  • the preselected members of the display library preselected set of potential binding partners
  • Such assays are thus preferably “inclusive” selecting for all binding partners rather than “exclusive” screening for a single one or few optimal binding partners.
  • the second screen is a conventional cDNA expression library screening method.
  • the expressed cDNA library is immobilized on a solid substrate (e.g. blotted onto a membrane) and then probed with the one or more targets.
  • Targets that specifically bind to the library members are identified and the binding members are optionally sequenced.
  • the target moieties are labeled with a detectable label.
  • Detectable labels suitable for use in the present invention include any composition detectable by spectroscopic, photochemical, biochemical, immunochemical, electrical, optical or chemical means.
  • Useful labels in the present invention include biotin for staining with labeled streptavidin conjugate, magnetic beads (e.g., DynabeadsTM), fluorescent dyes (e.g., fluorescein, texas red, rhodamine, green fluorescent protein, and the like, see, e.g., Molecular Probes, Eugene, Oregon, USA), radiolabels (e.g., 3 H, 125 1, 35 S, 14 C, or 32 P), enzymes (e.g.
  • a fluorescent label is preferred because it provides a very strong signal with low background. It is also optically detectable at high resolution and sensitivity through a quick scanning procedure.
  • the label can be coupled to the target moiety prior to, during, or after the binding assay.
  • directly labels are detectable labels that are directly attached to or incorporated into the target moiety prior to the binding assay.
  • indirect labels are joined to the target moiety/binding protein complex after binding.
  • the indirect label is attached to a second binding moiety that specifically binds to the target moiety or to a tag attached thereto.
  • the target moiety can be biotinylated before the screening assay.
  • an avidin-conjugated fluorophore will bind the biotin bearing complexes providing a label that is easily detected.
  • fluorescent labels are not to be limited to single species of organic molecules, but include inorganic molecules, multi-molecular mixtures of organic and/or inorganic molecules, crystals, heteropolymers, and the like.
  • CdSe-CdS core-shell nanocrystals enclosed in a silica shell can be easily
  • kits for the practice of the methods described herein include one or more components of a display library (e.g. phage display, bacterial display, yeast display, eukaryotic virus display library, direct plasmid display library, etc.) and instructional materials providing protocols for the assays disclosed herein.
  • a display library e.g. phage display, bacterial display, yeast display, eukaryotic virus display library, direct plasmid display library, etc.
  • instructional materials providing protocols for the assays disclosed herein.
  • instructional materials typically comprise written or printed materials they are not limited to such. Any medium capable of storing such instructions and communicating them to an end user is contemplated by this invention. Such media include, but are not limited to electronic storage media (e.g., magnetic discs, tapes, cartridges, chips), optical media (e.g., CD ROM), and the like. Such media may include addresses to internet sites that provide such instructional materials.
  • electronic storage media e.g., magnetic discs, tapes, cartridges, chips
  • optical media e.g., CD ROM
  • Such media may include addresses to internet sites that provide such instructional materials.
  • this invention contemplates the use of a database to permit storage, retrieval, and management of TAIS data.
  • a database can records showing amino acid sequence or identity of a member of a set of potential binding partners or proteins that interact with a one or more particular targets.
  • the term database refers to a means for recording and retrieving information. In preferred embodiments the database also provides means for sorting and/or searching the stored information.
  • the database can comprise any convenient media including, but not limited to, paper systems, card systems, mechanical systems, electronic systems, optical systems, magnetic systems or combinations thereof.
  • Preferred databases include electronic (e.g. computer-based) databases. Computer systems for use in storage and manipulation of databases are well known to those of skill in the art and include, but are not limited to
  • Results from screening of a T7 cDNA library derived from the normal human brain are presented and discussed below to demonstrate the potential of TAIS in mapping of protein-protein interactions.
  • SH3, PDZ and WW domains of the Abl, Src, Crk, PSD95 and Nedd4 proteins have been used as test targets.
  • 12 novel putative and 2 previously described interactions have been identified by TAIS for these well studied protein interaction modules.
  • TAIS when applied to cDNA libraries allows rapid and simultaneous exploration of combinatorial and natural peptide repertoires with protein interaction modules as targets. This feature makes TAIS an efficient tool for both direct mapping of protein-protein interactions and studies aiming to characterize molecular recognition properties of protein interaction modules.
  • a cDNA library derived from normal human brain was used in all presented screens (NOVAGEN. Cat. #70637-3. (2001), Novagen, Inc.).
  • the library was generated using purified poly(A) + mRNA from the brain tissue as a template to create first strand cDNAs, which in turn served as templates for the synthesis of double stranded cDNA fragments. In both cases priming was random, thus the size and composition of resultant cDNA inserts vary greatly.
  • the cDNA fragments longer than 300 base pair were directionally ligated to the C-terminus of gene product 10 of the lytic bacteriophage T7.
  • tissue-specific proteome is displayed on the surface of T7 phage as a C-terminal fusion to the major phage coat protein (NOVAGEN. OrienfExpress cDNA Manual, TB247. (1999)).
  • the reported diversities of tissue specific cDNA libraries from this source are in the order of 5xl0 7 primary recombinants, suggesting that even rare mRNA sequences are represented in these libraries with high probability (Soares et al. (1994) Proc. Natl. Acad. Sci, USA, 91: 9228-9232; Maniatis, et al. (1982) Molecular cloning. A Laboratory Manual, p. 225. (Cold Spring Harbor)).
  • ligand preferences of the tested domains have been characterized by screening of artificial peptide repertoires (Cheadle et al (1994) J Biol Chem 269: 24034-24039; Rickles et al. (1994) Embo J 13: 5598-5604; Sparks et al (1996) Proc. Natl. Acad. Sci, USA, 93: 1540-1544Sparks et al (1994) JBiol Chem 269: 23853-23856; Rickles et al. (1995) Proc. Natl Acad.
  • PDZ domains were originally described as 80-100 amino acid conserved repeats within the post-synaptic density 95 protein (PSD95) (Cho et al. (1992) Neuron 9: 929-942; Kornau et al (1997) Curr Opin Neurobiol 7: 368-373).
  • PSD95 post-synaptic density 95 protein
  • the prototypical PDZ domain protein PSD95 comprises three PDZ domains at the N-terminus followed by an SH3 domain and an inactive guanylate kinase domain (Cho et al. (1992) Neuron 9: 929- 942).
  • PDZ domains recognize and bind to the extreme C- terminal sequences of interacting partners with reported affinities from high nanomole to low micromole range (Niethammer et al. (1998) Neuron 20: 693-707; Songyang et al. (1997) Science 275: 73-77). Specificity of binding within the PDZ family is thought to be defined by 3-5 amino acids preceding the C-terminal residue (Songyang et al. (1997) Science 275: 73-77; Strieker et al (1997) Nat Biotechnol 15: 336-342; Doyle et al.
  • a cDNA human brain library displayed on the T7 phage was TAISed with the N-terminal fragment of PSD95 comprising three PDZ domains as a target (PSD95- PDZ(l+2+3)).
  • the pre-selected cDNA library formed about 1500 plaques on a bacterial lawn, when plated on two 150 mm Petri dishes. 11 clones gave positive signals on the membranes after plaque lift and screening of membranes with biotinylated PSD95- PDZ(l+2) complexed to streptavidin-alkaline phophatase (AP) conjugate (see Fig. 2).
  • Table 1 Results of screening of a phage-displayed human brain cDNA library with an N-terminal fragment of PSD95 comprising its three PDZ domains. Sequences of polypeptides displayed by phages from positive plaques along with their relative affinity ranks towards the target and identities of the respective cDNA inserts. FS frameshift, ? - undefined, DGK ⁇ - diacylglycerol kinase zeta, UTR - untranslated region, ">" - denotes free carboxylate group.
  • the minimum consensus sequence of peptides that bound PSD95-PDZ(l+2) can be readily defined as (R/K)-x-(S/T)-x-(V/T)-COOH (SEQ ID NO: 16).
  • This consensus matches well with C-terminal sequences of known interacting partners of PSD95, such as inward rectifier K + channel (Kir2.3: NISYRRESAI-COOH, SEQ ID NO: 17) (Cohen et al. (1996) Neuron 17: 759-767), embryonic skeletal muscle sodium channel (SkM2: SPDRDRESLV-COOH, SEQ ID Nolle) (Gee et al.
  • the cDNA library can be viewed as a combinatorial library that is highly enriched in natural peptide sequences.
  • the latter provide a unique internal reference about physiologically relevant affinities and specificities when the library is assayed for the interaction with a target protein.
  • PDl and PD2 peptides that bound strongly to PSD95-PDZ(l+2+3), may represent novel proteins that interact with PSD95.
  • the nucleotide sequences of PDl and PD2 inserts match a number of human ESTs and genomic sequences with no assigned open reading frame (not shown). The biochemical characterization of corresponding full-length cDNA products can substantiate this putative activity/function.
  • Kinesin-like protein KIF1B [Mus musculus (Mouse) ] SPLICE ISOFORM 3 OF Q60575 1143-1150
  • Kinesin-like protein Kiflb alpha [Brachydanio rerio (Zebrafish) (Danio rerio) ] . 1154-1161
  • Trans-activating transcriptional regulatory KHFRETEV 31 protein (X-LOR protein) (PX protein) .
  • PX protein Trans-activating transcriptional regulatory KHFRETEV 31 protein
  • HTLV-I Human T-cell leukemia virus type I (strain ATK & Caribbean isolate) 351-358
  • Human papillomavirus type 45 (conforms for types 56, 68, 70, ME180, 151-158
  • GAG polyprotein [Contains: core protein (s)
  • RLIP76 protein (Similar to ralA binding protein 1) .
  • Xenopus laevis Africann clawed frog 604-611
  • Diacylglycerol kinase, zeta (EC 2.7.1.107) REDQETAV 43 Diglyceride kinase) (DGK-zeta) (DAG kinase zeta) [Homo sapiens (Human) ] . 1110-1117
  • Diacylglycerol kinase, zeta (EC 2.7.1.107) (Diglyceride kinase) (DGK-zeta) (DAG kinase zeta) (DGK-IV) (104 kDa diacylglycerol kinase) [Rattus norvegicus (Rat) ] . 922-929
  • Q9VHT6 CG9626 protein [Drosophila melanogaster (Fruit fly) ] .
  • PSD95-binding sequences at the C-termini of proteins from different Chlamydia strains may indicate on interesting and unexpected molecular connections exploited by this intracellular parasite, which is implicated in a host of human ailments such as trachoma, arthritis, Alzheimer's disease among others.
  • Figure 3 illustrates another example of PDZ domain profiling.
  • the x-axis shows an array of individual phages selected to bind a number of different PDZ domains, while the y-axis shows the relative affinities of individual phages to the 2nd PDZ domains
  • Table 4 illustrates PDZ2 domain best binders.
  • SAP97_PDZ2 domain best binders SEQ ID NO: 1
  • DGK ⁇ Diacylglycerol kinase zeta
  • DAG lipid second messenger diacylglycerol
  • DAG is generated by phosphoinositide-specific phospholipase C (PLC) isoforms and accumulates locally and transiently upon activation of a large number of growth factor and other cell surface receptors (Bishop and Bell (1986) J Biol Chem 261: 12513-12519; Rhee (2001) Annu Rev Biochem 70: 281-312).
  • PLC phosphoinositide-specific phospholipase C
  • WW domains are protein interaction modules recognizing short proline-rich sequences (Bork and Sudol (1994) Trends Biochem Sci 19: 531-533). They are found in proteins with functions as diverse as cell cycle control, pre-mRNA 3' end formation and targeted protein degradation (Sudol and Hunter (2000) Cell 103: 1001-1004; Lu et al. (1999) Science 283: 1325-1328; Morris et al (1999) JBiol Chem 274: 31583-31587; Morris and Greenleaf (2000) JBiol Chem 275: 39935-39943; Verdecia et al. (2000) Nat Struct Biol 1: 639-643).
  • WW domains are segregated into at least five classes (Kasanov et al. (2001) Chem Biol 8: 231-241): Class I prefers peptide ligands with a core motif PPxY (Chen and Sudol (1995) Proc. Natl. Acad. Sci, USA, 92: 7819-7823); Class II - PPLP (Bedford et al. (1997) Embo J 16: 2376-2383); Class HI - PxxGMxxPP (Bedford et al. Proc. Natl Acad. Sci, USA, 95: 10602-10607); Class IV - (pS/pT)P (Lu et al. (1999) Science 283: 1325-1328); and Class V - RxPPGPPPxR (Komuro et al (1999) JBiol Chem 274: 36513-36519).
  • the Nedd4-WW3 belongs to the Class I WW domains and a characteristic Class I core recognition motif PPxY is readily discernible in all selected peptide sequences (underlined in Table 5).
  • PPAYGRG SEQ ID NO:75
  • PPPYPTP SEQ ID NO:73
  • the chimaerin homologue may be a false positive picked up due to avidity provided by two closely situated PPxY core motifs.
  • Nedd4 has been proposed to control stability and/or turnover of ENaC at the cell surface, presumably by directing its ubiquitination, which is followed by endocytosis and degradation of the channel (Staub et al. (1996) Embo J 15: 2371-2380; Staub et al (1997) Embo J 16: 6325-6336; Abriel et al. (1999) / Clin Invest 103: 667-673).
  • WW domains of Nedd4 are thought to function in this system as targeting modules, since they specifically bind subunits of ENaC.
  • Deletions or point mutations in the PPxY motif on ⁇ or ⁇ subunits of ENaC are associated with a hereditary form of hypertension, Liddle's syndrome, which is characterized by deregulated activity of ENaCs (Shimkets et al (1994) Cell 79: 407-414.
  • Nedd4 and Nedd4-like proteins due to their unique structure comprising a membrane targeting C2 domain, two to four WW domains and a C-terminal HECT -type ubiquitin protein ligase domain, are strong candidates for regulators of ubiquitin-mediated turnover of many membrane proteins (JoUiffe et al (2000) Biochem J 351 Pt 3, 557-565; Abriel et al. (2000) FEBS Lett 466: 377- 380; Rotin et al. (2000) J Membr Biol 176: 1-17).
  • the yeast ubiquitin-protein ligase Rsp5p a homologue of mammalian Nedd4 and Itch, is required for the ubiquitination and subsequent internalization of several plasma membrane proteins, including the alpha-factor receptor (Ste2p) (Hicke et al (11996) Cell 84: 277-287; Dunn and Hicke (2001) Mol Biol Cell 12: 421-435), uracil permease (Galan et al. (1996) JBiol Chem 271: 10946-10952), general amino acid permease (Springael et al. (1998) Mol Biol Cell 9: 1253-1263) and others (Hicke (1997) Faseb i ll: 1215-1226). Therefore, it is reasonable to assume an existence of multiple Nedd4 targets in the cell.
  • Step2p the alpha-factor receptor
  • uracil permease Galan et al. (1996) JBiol Chem 271: 10946-10952
  • Nogo-A, lysosomal-associated multispanning membrane protein 5 (LAPTM5), type JJ ⁇ subunit of voltage gated sodium channel (SCN2A) and a novel human protein with homology to chimaerin have been identified by TAIS as novel putative
  • Nogo-A has been recently cloned independently by three different teams as a long sought myelin inhibitor of regenerating axons, and is the subject of intensive studies assessing the contribution of Nogo to the failure of axonal regeneration in the adult CNS (Prinjha et al. (2000) Nature 403: 383-384; GrandPre et al. (2000) Nature 403: 439-444; Chen et al. (2000) Nature 403: 434-439).
  • a possible regulation of Nogo-A through ubiquitin-mediated degradation pathways may provide a fruitful framework for studies aiming to understand the molecular basis of CNS regeneration and plasticity.
  • LAPTM5 was originally cloned as a lysosomal membrane associated protein that interacts with ubiquitin, developmentally downregulated and preferentially expressed in adult tissues with high cell turnover (Adra et al (1996) Genomics 35: 328-337). The function of the protein is unknown.
  • the rat homologue of mouse LAPTM5, Granule Cell Death - 10 protein (GCD-10), is up-regulated in microglia in response to degeneration and cell death of neurons in vitro and in vivo and is involved in the dynamics of lysosomal membranes of activated microglia (Origasa et al. (2001) Brain Res Mol Brain Res 88: 1-13).
  • VGSC voltage-gated sodium channels
  • Table 6 shows results of screening of a human brain cDNA library with the third WW domain of Nedd4 ubiquitin ligase as a target. Homologous sequences shared by polypeptides selected with Nedd4-WW3 domain as defined by the BLOCK MAKER algorithm (see, e.g., http://www.blocks.fhcrc.org/blockmkr/make_blocks.html).
  • Table 7 shows results of screening of a human brain cDNA library with the third WW domain of Nedd4 ubiquitin ligase as a target.
  • PPxYESL SEQ ID NO:85, Kay et al.
  • the PPSYDSV (SEQ ID NO: 102) sequence: i) is strictly conserved across species and between different alpha subunit isoforms of cardiac and neuronal VGSCs; ii) is embedded in sequences shown to be prerequisite for proteins degraded through ubiquitin-directed endocytosis, such as PEST sequences, multiple serines and threonines (phosphorylation acceptors) and lysines (ubiquitination acceptors); and iii) conforms well to recognition consensus of the Nedd4 WW3 domain, PPxYES(L/M) (SEQ ID NO: 103), defined recently by a combinatorial peptide library approach (Kay et al.
  • Nedd4 ubiquitin-protein ligase (Abriel et al. (2000) FEBS Lett 466: 377-380)
  • strict conservation of the Nedd4-WW3 recognition sequence within C-termini of cardiac and neuronal voltage gated sodium channels and an in vitro interaction of Nedd4-WW3 with a C-terminus of alpha subunit of neuronal VGSC (as noted in the present paper) strongly suggest a role of the Nedd4 ubiquitin-mediated endocytotic pathway in the regulation of stability and/or turnover of neuronal VGSC. It is relevant that high expression of Nedd4 was demonstrated in the heart and nervous tissues (Staub et al. (1996) Embo J 15: 2371-2380).
  • RhoGAP domain GTPase activators for Rho-like GTPases
  • SH3 domain The Src homology 3 (SH3) domain has become a prototype of protein interaction modules since it was first described as a conserved repeat in the N-terminus of Src family tyrosine kinases (Koch et al. (1991) Science 252: 668-674). Small, about 50-70 amino acids long, with a compact fold, SH3 domains recognize and bind peptide sequences with the core PxxP motif. The specificity of interaction within the SH3 family is determined by additional contacts formed between amino acids adjacent to the PxxP core of peptide ligand and variable amino acids within SH3 domain specificity pocket (Rickles et al. (1995) Proc. Natl. Acad.
  • Peptide ligands can bind SH3 domains in two pseudosymmetrical (with respect to the PxxP core motif) orientations - the Class I orientation, ZxxPxxP, and the Class II orientation, PxxPxZ, where Z denotes the ligand residue(s) responsible for discrimination between individual SH3 domains (Feng et al (1994) Science 266: 1241-1247).
  • SH3 domains within the Src and Abl tyrosine kinases are believed to be two-fold.
  • SH3 domains of Src and Abl participate in the autoinhibitory control of the respective kinases (Sicheri and Kuriyan (1997) Curr Opin Struct Biol 7: 777-785; Barila and Superti-Furga (1988) Nat Genet 18: 280-282).
  • they serve as targeting modules by binding to a specific subset of proteins containing polyproline sequences (Koch et al (1991) Science 252: 668- 674; Pawson and ⁇ ash (2000) Genes Dev 14: 1027-1047). Therefore, identification of binding partners of SH3 domains of the tyrosine kinases either directly suggests physiological targets of their activity or may indicate the multiprotein complexes to which they are targeted.
  • Crk is an adaptor protein composed of an SH2 domain and one or two (depending on the isoform) SH3 domains (Feller et al. (1998) J Cell Physiol 111: 535-552). By interacting with specific sets of proteins via their interaction modules, adaptor proteins
  • -41- function to provide a molecular connection between signal transduction pathways. Identification of interaction partners of an adaptor protein facilitates the unraveling of interconnections and possible cross-talk between different signaling cascades.
  • c-Src and c-Abl tyrosine kinases and the adaptor protein Crk are cellular counterparts of classical viral oncogenes, v-Src (Radke et al. (1980) Cell 21: 821-828), v- Abl (Rosenberg and Witte (1988) Adv Virus Res 35: 39-81) and v-Crk (Mayer et al (1988) Nature 332: 272-275).
  • the pathways affected by these oncogenes have been the subjects of extensive studies with a number proteins identified as interacting partners of the respective SH3 domains (Barfod et al (1993) JBiol Chem 268: 26059-26062; Weng et al. (1994) Mol Cell Biol 14: 4509-4521; Kapeller et al (1994) JBiol Chem 269: 1927-1933; Gout et al
  • Table 8 Summary of TAIS performed on a phage-displayed human brain cDNA library with the indicated targets.
  • Table 9 Alignments of polypeptides selected from a phage-displayed human brain cDNA library by the indicated SH3 domains in comparison to previously reported recognition consensuses of corresponding SH3 domains. Underlined residues in previously reported consensuses for Src and Crk SH3 domains are position that have been fixed in biased peptide libraries used to define the respective consensuses, ⁇ denotes aliphatic residues. Note the additional specificity determinants uncovered by TAIS for the Crk SH3 domain at +4 and +5 positions (in respect to the PxxP core) of the selected peptide ligands.
  • ⁇ is aromatic residue
  • a significant fraction of all specific protein-protein associations in the cell may be mediated by specialized peptide recognition domains such as PDZ, SH3, WW, EH, SH2, etc. Indeed, 3300 proteins out of 6148 predicted ORFs in the yeast proteome have been reported to contain the SH3 domain recognition core PxxP (Zucconi et al. (2000) FEBS Lett 480: 49-54; Cherry et al. (1998) Nucleic Acids Res 26: 73-79). Similarly, SH3 and PDZ domains were ranked as 14 th and 19 th , respectively, among the most populous domain families in the human proteome (Lander et al. (2001) Nature 409: 860-921). On the qualitative side, protein interaction modules, in the context of proteins with enzymatic,
  • TAIS in vitro method
  • Immobilized GST fusions of target proteins were purified according to the supplier's instructions (Pharmacia Biotech.).
  • STRAP streptavidin-alkaline phosphatase conjugate
  • target domains were released from Glutathione Sepharose 4B beads by thrombin cleavage and mixed with freshly prepared water solution of EZ-linkTM Sulfo-NHS-LC-LC-biotin (Pierce) at a molar ratio of 1:5. Biotinylation reaction was incubated for 30 minutes at room temperature followed by purification on MicroSpin G-25 column (Pharmacia Biotech.).
  • biotinylation was kept at 1 to 2 moieties of biotin per target molecule.
  • 5 ⁇ g of biotinylated target per membrane were pre-mixed with STRAP conjugate at a molar ratio of 4: 1 to ensure multivalent target presentation and incubated for 10 minutes at RT before use in Tris-buffered saline, pH7.4 + 0.1% Tween 20 (TBS-T).
  • -48- plaques on membranes were developed with insoluble AP substrate, BCIP/NBT (Sigma). Individual positive plaques were identified on plates and picked up for sequencing. If density of plaques was too high to pick up individual phage, agar stubs containing positive plaques were excised and phages from stubs eluted in PBS. Eluted phages were plated for a secondary screening on membranes. T7 phage DNA was prepared for sequencing with lambda DNA Wizard kit from Promega.

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Publication number Priority date Publication date Assignee Title
WO1996009411A1 (en) * 1994-09-21 1996-03-28 Cytogen Corporation Antigen binding peptides (abtides) from peptide libraries
WO2000071694A1 (en) * 1999-05-25 2000-11-30 The Scripps Research Institute METHODS FOR DISPLAY OF HETERODIMERIC PROTEINS ON FILAMENTOUS PHAGE USING pVII and pIX, COMPOSITIONS, VECTORS AND COMBINATORIAL LIBRARIES

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US5733731A (en) * 1991-10-16 1998-03-31 Affymax Technologies N.V. Peptide library and screening method
US5702892A (en) * 1995-05-09 1997-12-30 The United States Of America As Represented By The Department Of Health And Human Services Phage-display of immunoglobulin heavy chain libraries
US6031071A (en) * 1996-01-24 2000-02-29 Biophage, Inc. Methods of generating novel peptides
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* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1996009411A1 (en) * 1994-09-21 1996-03-28 Cytogen Corporation Antigen binding peptides (abtides) from peptide libraries
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Non-Patent Citations (9)

* Cited by examiner, † Cited by third party
Title
DE WILDT RUUD M T ET AL: "Antibody arrays for high-throughput screening of antibody-antigen interactions", NATURE BIOTECHNOLOGY, NATURE PUBLISHING GROUP, NEW YORK, NY, US, vol. 18, no. 9, September 2000 (2000-09-01), pages 989 - 994, XP002162316, ISSN: 1087-0156 *
FUH G ET AL: "Analysis of PDZ domain-ligand interactions using carboxyl-terminal phage display", JOURNAL OF BIOLOGICAL CHEMISTRY, AMERICAN SOCIETY OF BIOLOCHEMICAL BIOLOGISTS, BIRMINGHAM,, US, vol. 275, no. 28, 14 July 2000 (2000-07-14), pages 21486 - 21491, XP002356319, ISSN: 0021-9258 *
KAY B K ET AL: "Convergent evolution with combinatorial peptides", FEBS LETTERS, ELSEVIER, AMSTERDAM, NL, vol. 480, no. 1, 25 August 2000 (2000-08-25), pages 55 - 62, XP004597873, ISSN: 0014-5793 *
KAY B K ET AL: "From peptides to drugs via phage display", DRUG DISCOVERY TODAY 1998 UNITED KINGDOM, vol. 3, no. 8, 1998, pages 370 - 378, XP002405768, ISSN: 1359-6446 *
KURAKIN ALEXEI ET AL: "Target-assisted iterative screening reveals novel interactors for PSD95, Nedd4, Src, Abl and Crk proteins", JOURNAL OF BIOMOLECULAR STRUCTURE AND DYNAMICS, vol. 19, no. 6, June 2002 (2002-06-01), pages 1015 - 1029, XP009074465, ISSN: 0739-1102 *
PINCUS S ET AL: "Peptides that mimic the group B streptococcal type III capsular polysaccharide antigen", JOURNAL OF IMMUNOLOGY, THE WILLIAMS AND WILKINS CO. BALTIMORE, US, vol. 160, no. 1, 1998, pages 293 - 298, XP002102148, ISSN: 0022-1767 *
SALCINI A E ET AL: "BINDING SPECIFICITY AND IN VIVO TARGETS OF THE EH DOMAIN, A NOVEL PROTEIN-PROTEIN INTERACTION MODULE", GENES AND DEVELOPMENT, COLD SPRING HARBOR, NY, US, vol. 11, no. 17, 1 September 1997 (1997-09-01), pages 2239 - 2249, XP002073498, ISSN: 0890-9369 *
See also references of WO03029821A1 *
SPARKS A B ET AL: "CLONING OF LIGAND TARGETS: SYSTEMATIC ISOLATION OF SH3 DOMAIN- CONTAINING PROTEINS", NATURE BIOTECHNOLOGY, NATURE PUBLISHING GROUP, NEW YORK, NY, US, vol. 14, no. 6, June 1996 (1996-06-01), pages 741 - 744, XP000876847, ISSN: 1087-0156 *

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