WO2008140538A1 - Ecran d'affichage d'adn pour un produit d'expression avec des propriétés de liaison désirées - Google Patents

Ecran d'affichage d'adn pour un produit d'expression avec des propriétés de liaison désirées Download PDF

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Publication number
WO2008140538A1
WO2008140538A1 PCT/US2007/080350 US2007080350W WO2008140538A1 WO 2008140538 A1 WO2008140538 A1 WO 2008140538A1 US 2007080350 W US2007080350 W US 2007080350W WO 2008140538 A1 WO2008140538 A1 WO 2008140538A1
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nucleic acid
fusion protein
acid molecule
molecule
nam
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PCT/US2007/080350
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Jeff Rogers
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Verenium Corporation
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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
    • 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
    • C12N15/1055—Protein x Protein interaction, e.g. two hybrid selection

Definitions

  • the present invention relates generally to methods of screening polypeptides for binding to a ligand or other target molecule of interest, and more particularly to methods for generating and screening large polypeptide libraries for polypeptides with desired binding characteristics.
  • the present invention provides a method for isolating a nucleic acid sequence encoding a polypeptide that binds to a target molecule (TM).
  • the method comprises (a) providing a first nucleic acid molecule-fusion protein complex (1st NAM:NABD-CP), (b) providing a second nucleic acid molecule-fusion protein complex (2nd NAM:NABD-CP), (c) contacting the complexes with the target molecule to form a target- molecule-containing complex ((NAM:NABD-CP) 2 :TM), and (d) isolating nucleic acids from this complex.
  • TM target- molecule-containing complex
  • a nucleic acid molecule has a target sequence of a nucleic- acid-binding domain (NABD-TS) and a sequence encoding a fusion protein.
  • the fusion protein contains a nucleic-acid-binding domain (NABD) and a candidate polypeptide (CP).
  • the NABD of the fusion protein can bind to the target sequence of the nucleic-acid-binding domain (NABD-TS).
  • the NAM of step (a) is bound to the fusion protein to provide the "first nucleic acid molecule-fusion protein complex" (1st NAM:NABD-CP).
  • a similar 2nd NAM:NABD-CP is provided in step (b).
  • the invention also provides a method of (a) providing a nucleic acid molecule-fusion protein complex (NAM: ZF -CP), (b) contacting the NAM:ZF-CP with a target molecule, forming a target-molecule-containing complex (NAM:ZF-CP:TM); and (c) isolating a nucleic acid molecule of the NAM:ZF-CP:TM complex.
  • the nucleic acid molecule has a Zn-finger target sequence (ZF-TS) and the fusion protein contains a Zn-finger-binding domain (ZF).
  • Figure 1 provides a schematic of the DNA Display method provided herein.
  • Figure 2 provides a schematic of the Assembly DNA Display method provided herein.
  • Figure 3 depicts ELISA assay results from members of a library screened for SARS spike protein binding according to the DNA Display methods provided herein.
  • Figure 4 depicts ELISA assay results of the specificity of hits from a library screened for lysozyme binding according to the Assembly DNA Display methods provided herein.
  • Figure 5 depicts the nucleic acid and amino acid sequences of the Zn- finger binding domain, as well as His and Flag tags.
  • Figure 6 to Figure 9 depict maps of vectors pBAD ZF, pBAD33_ZF, pBAD_ZF_3889Fab33, and pBAD_ZF_3889Fab35
  • the present application is directed to methods for screening candidate polypeptides to identify compounds that bind to a target molecule of interest (or ligand). Also provided are polypeptide libraries that can be used in these methods.
  • the candidate polypeptides are produced from polypeptide expression vectors that encode the candidate polypeptides attached to a DNA-binding protein.
  • the vector encoding the candidate polypeptide-encoding gene is constructed so that the DNA binding protein-candidate polypeptide fusion product can bind to the recombinant DNA expression vector that encodes the fusion product containing the candidate polypeptide.
  • the present methods of ligand-binding selection allow a very large library of candidate polypeptides to be screened, and any vector(s) encoding a candidate polypeptide(s) with the desired binding properties to be selected.
  • the vector can then be isolated and further screened, and/or sequenced to deduce the amino acid sequence of the candidate polypeptide with the desired binding properties.
  • Using these methods one can identify a polypeptide as having a desired binding affinity for a target molecule.
  • the polypeptide can then be synthesized in bulk by conventional means.
  • nucleic acid molecule or simply "nucleic acid” refers to a polymeric molecule comprising nucleotide units.
  • An NAM such as a genomic DNA, cDNA or mRNA, can encode or be translated to express a polypeptide, such as a fusion protein.
  • a fusion protein refers to two or more polypeptide chains from different proteins that are produced as a single polypeptide product. In some cases, the different proteins are produced as a result of a lack of a stop codon between the two or more polypeptide chains.
  • the two or more polypeptide chains can be entire polypeptides or fragments thereof, for example, an antibody fragment.
  • a candidate polypeptide refers to a polypeptide for which its binding ability to a target molecule is to be determined.
  • a candidate polypeptide can be a full-length protein or a fragment thereof, or a sequence variant thereof.
  • the candidate polypeptide can be expressed alone or as a portion of a fusion protein (e.g., a fusion protein with an NABD).
  • An exemplary candidate polypeptide is an active fragment of an antibody.
  • a target molecule refers to a molecule to which binding of a polypeptide is to be developed using the methods provided herein.
  • a target molecule can be a small molecule, such as a drug, a sugar, or other organic molecule, or can be a large biomolecule such as a protein, polysaccharide or polynucleotide.
  • a target-molecule-containing complex refers to a complex of at least a TM and a candidate polypeptide bound thereto. Additional components of a TMCC can include an nucleic-acid-binding domain with which the candidate polypeptide is associated, a second candidate polypeptide or fusion protein thereof, and one or more NAMs encoding the one or more candidate polypeptides bound to the TM.
  • exemplary target-molecule-containing complexes are NAM:ZF-CP:TM in Figure 1 and (NAM:NABD-CP) 2 :TM in Figure 2.
  • bind, bound and binding refer to the binding between atoms or molecules with a K d in the range of 10 "2 to 10 "15 mole/L, generally, 10 "6 to 10 "15 , 10 “7 to 10 “15 and typically 10 "8 to 10 "15 mole/L (and/or a K a of 10 5 -10 12 , 10 7 -10 12 , 10 8 -10 12 L/mole).
  • specific binding of a first compound to a second compound is a level of binding having at least about 2-fold and typically at least about 5-fold, 10-fold, 50- fold, 100-fold, or more, greater affinity (K a or K eq ) than for another molecule (e.g., a random or negative control molecule such as lysozyme, BSA or milk protein for protein binding or salmon sperm DNA for nucleic acid molecule binding), or at least 2-fold and typically at least 5-fold, 10-fold, 50-fold, 100-fold, or more, greater affinity (K a or K eq ) than for another molecule.
  • Typical conditions for detecting and determining binding affinity constants or for determining the selectivity of binding include physiological conditions, such as PBS (137 mM NaCl, 2.7 mM KCl, 10 mM phosphate buffer pH 7.4).
  • association with or “associates with” refers to the multimerization of biomolecules, such as, for example, dimerization of proteins.
  • associated proteins include, but are not limited to, association between antibody heavy and light chains, and coiled-coil multimerization (e.g., GCN4 leucine zipper dimer or gp41 hexamer).
  • contacting refers to a process of adding, mixing or otherwise bringing into association two or more components.
  • a candidate polypeptide is contacted with a target molecule when a solution containing the candidate polypeptide is admixed with a solution containing the target molecule.
  • isolation refers to separating a compound from its natural environment, or from one or more impurities.
  • a candidate polypeptide that binds a TM can be isolated by separating a TMCC from candidate polypeptides not bound to the TM.
  • Exemplary, non-limiting, isolating methods include immunoprecipitation, magnetic bead harvesting, and cell sorting, where isolating methods can be accompanied by one or more washing steps.
  • sequencing refers to elucidating the identity of nucleotides in at least a portion of a nucleic acid molecule and/or elucidating the identity of amino acids in at least a portion of a polypeptide or protein.
  • Various methods for sequencing polynucleotides and amino acids are known in the art, including, but not limited to, dideoxy- based nucleotide sequencing and mass spectroscopy amino acid sequencing.
  • at least a portion refers to the fact that the methods provided herein may include, but do not require that the entire nucleotide or amino acid sequence of a biomolecule be elucidated. For example, when an Fab light chain is used as a candidate polypeptide, the sequencing can be performed on only the variable domain portion of the Fab light chain.
  • NABD nucleic-acid-binding domain
  • a NABD binds to a particular nucleotide sequence of a nucleic acid molecule.
  • TS target sequence of an NABD refers to the particular nucleotide sequence of a nucleic acid molecule to which the NABD binds.
  • ZF Zn-finger-binding domain
  • NABD NABD that contains one or more particular three-dimensional structural motifs known in the art as Zn- fingers. Each of such motifs typically contain a Zn atom coordinated by at least three amino acids, more typically at least four amino acids, which are typically cysteine or histidine.
  • An exemplary ZF is provided in SEQ ID NO:2, as encoded by SEQ ID NO: 1.
  • ZF-TS Zn-finger target sequence
  • a "library” refers to a plurality of different but related members that can be screened for members having desired properties.
  • a peptide library can be a plurality of different peptides having a designated range of numbers of amino acids (e.g., 15-50 amino acids), and an antibody library can be a plurality of different antibodies, heavy or light chains, or fragments thereof.
  • an "immunoassay” is defined as any method using a specific or preferential binding of an antigen with a second material (i.e., a binding partner, usually an antibody, antibody fragment or another substance having an antigen binding site) that specifically or preferentially binds to an epitope of the antigen.
  • a binding partner usually an antibody, antibody fragment or another substance having an antigen binding site
  • the immunoassay methods provided herein include any known to those of skill in the art, including, but not limited to, ELISA, sandwich, competition, agglutination, or precipitation assays.
  • antibody refers to an immunoglobulin, including any derivative or fragment thereof that retains the specific binding ability of the antibody.
  • An active fragment of an antibody refers to a portion of an antibody that retains the specific binding ability of the antibody.
  • antibody or active fragment thereof includes any protein having an immunoglobulin binding domain or a binding domain that is homologous or substantially homologous to an immunoglobulin binding domain.
  • the term antibody includes antibody fragments, such as Fab fragments, which comprise a light chain and the variable region of a heavy chain.
  • Antibodies include members of any immunoglobulin class, including IgG, IgM, IgA, IgD and IgE.
  • an "antibody fragment” refers to any derivative of an antibody that is less than a full length antibody, retaining at least a portion of the full-length antibody's specific binding ability.
  • antibody fragments include, but are not limited to, Fab, Fab', F(ab) 2 , single-chain Fvs (scFv), small immune proteins, Fv, dsFv diabody and Fd fragments.
  • the fragment can include multiple chains linked together, such as by disulfide bridges.
  • An antibody fragment generally contains at least about 50 amino acids and typically at least about 200 amino acids, or at least 50 amino acids and typically at least 200 amino acids.
  • a Fab fragment is an antigen-binding antibody fragment containing one variable heavy domain (V H ), one variable light (V L ) domain, one constant heavy domain 1 (C HI ) and one constant light (C L ) domain.
  • a Fab heavy chain is an antigen-binding antibody fragment containing one variable heavy domain (V H ) and one constant heavy domain 1 (C HI ).
  • a Fab light chain is an antigen-binding antibody fragment containing one variable light (V L ) domain and one constant light (C L ) domain.
  • hsFv refers to antibody fragments in which the constant domains normally present in an Fab fragment have been substituted with a heterodimeric coiled-coil domain (see, e.g., Arndt et al. J. MoI. Biol. 7:312:221-228 (2001)).
  • an F(ab) 2 fragment is an antibody fragment containing two variable heavy domains (V H ), two variable light (V L ) domains, two constant heavy domains 1 (C HI ) and two constant light (C L ) domains.
  • a Fv antibody fragment is composed of one variable heavy domain (V H ) and one variable light (V L ) domain linked by non-covalent interactions.
  • a dsFv refers to a Fv with an engineered intermolecular disulfide bond, which stabilizes the V H -V L pair.
  • scFvs refer to antibody fragments that contain a variable light chain domain (V L ) and variable heavy chain domain (V H ) covalently connected by a polypeptide linker in any order. The linker is of a length such that the two variable domains are bridged without substantial interference.
  • linkers are (Gly-Ser) n residues with some GIu or Lys residues dispersed throughout to increase solubility.
  • diabodies are dimeric scFv; diabodies typically have shorter peptide linkers than scFvs and they preferentially dimerize.
  • small immune proteins SIP are scFv fragments connected to a dimerization domain of an antibody, such as an IgG CH 3 domain.
  • autoantibody refers to an antibody produced by a subject that binds to an endogenous antigen of the subject.
  • an autoantibody can be produced in response to presence of a tumor, cancer, or cancerous condition with the subject.
  • Autoantibodies although produced by the subject in response to an endogenous antigen, can be detected or measured by reaction of the autoantibody with a binding partner, such as a test antigen produced or obtained from a variety of sources including by recombinant techniques.
  • amplify, amplified and amplifying refer to methods for increasing the number of copies of a specific nucleic acid molecule, such as a DNA fragment.
  • amplify, amplified and amplifying include in vitro processes wherein a nucleic acid molecule is increased in copy number using known techniques such as, for example, cloning, transcription, the polymerase chain reaction (PCR), the ligase chain reaction (LCR) and strand displacement, and in vivo processes in which nucleic acid molecules replicate within a cell.
  • a detectable label or detectable moiety refers to an atom, molecule or composition, wherein the presence of the atom, molecule or composition can be directly or indirectly measured.
  • a label can be detected, for example, by visual inspection, by fluorescence spectroscopy, by reflectance measurement, by flow cytometry, or by mass spectrometry.
  • Direct detection of a detectable label refers to measurement of a physical phenomenon, such as energy or particle emission or absorption, of the moiety itself.
  • Indirect detection refers to measurement of a physical phenomenon, such as energy or particle emission or absorption, of an atom, molecule or composition that binds directly or indirectly to the detectable moiety.
  • a detectable label can be biotin, which can be detected by binding to avidin and avidin can be detected by, for example, binding avidin with a second biotin molecule linked to fluorescein.
  • a detectable label or detectable moiety included within the scope of a detectable label or detectable moiety is a bindable label or bindable moiety, which refers to an atom, molecule or composition, wherein the presence of the atom, molecule or composition can be detected as a result of the label or moiety binding to another atom, molecule or composition.
  • an “isolatable moiety” refers to an atom, molecule or composition, wherein the presence of the atom, molecule or composition can be bound, segregated or otherwise separated from other components of a mixture.
  • Materials that can be used for an isolatable moiety include any material that can be used as affinity matrices, supports or beads for chemical and biological molecule syntheses and analyses, such as, but are not limited to: organic or inorganic polymers, biopolymers, natural and synthetic polymers, including, but not limited to, agarose, cellulose, nitrocellulose, cellulose acetate, other cellulose derivatives, dextran, dextran-derivatives and dextran co-polymers, other polysaccharides, gelatin, polyvinyl pyrrolidone, rayon, nylon, polyethylene, polypropylene, polybutylene, polycarbonate, polyesters, polyamides, vinyl polymers, polyvinylalcohols, polyvinylidenedifluoride (
  • CPG controlled-pore glass
  • silica gels silica gels, ceramics, paper, natural sponges, insoluble protein, surfactants, red blood cells, metals (including metal ions; e.g., steel, gold, silver, aluminum and copper), metalloids, magnetic materials (including TeflonTM-coated magnetic materials and magnetic beads), Wang resin, Merrifield resin, SephadexTM, SepharoseTM, nylon, dextran, chitin, sand, pumice, dendrimers, buckyballs, or other commercially available medium.
  • CPG controlled-pore glass
  • Exemplary supports include, but are not limited to flat supports such as glass fiber filters, silicon surfaces, glass surfaces, latex beads, magnetic beads, nitrocellulose membranes, tissue culture plates, microarrays, metal surfaces (steel, gold, silver, aluminum and copper) and plastic materials. Screening Methods
  • the present application is directed to methods for screening candidate polypeptides to identify compounds that bind to a target molecule of interest.
  • polypeptide libraries that can be used in these methods.
  • the candidate polypeptides are produced from polypeptide expression vectors that encode the candidate polypeptides attached to a DNA binding protein.
  • the vector encoding the candidate polypeptide-encoding gene is constructed so that the DNA binding protein-candidate polypeptide fusion product can bind to the recombinant DNA expression vector that encodes the fusion product containing the candidate polypeptide.
  • the present methods of ligand binding selection allows a very large library of candidate polypeptides to be screened and any vector(s) encoding a candidate polypeptide(s) with the desired binding properties can be selected.
  • the vector can then be isolated and further screened, and/or sequenced to deduce the amino acid sequence of the candidate polypeptide with the desired binding properties.
  • Using these methods one can identify a polypeptide as having a desired binding affinity for a target molecule.
  • the polypeptide can then be synthesized in bulk by conventional means.
  • nucleic acid sequence encoding a polypeptide that binds to a target molecule comprising the steps of: (a) providing a nucleic acid molecule-fusion protein complex comprising: (i) a nucleic acid molecule (NAM) comprising a target sequence (TS) of a nucleic-acid-binding domain (NABD) and further comprising a sequence encoding a fusion protein that contains a NABD and a candidate polypeptide (NABD-CP), wherein the NABD is capable of binding to the TS of the NABD; and (ii) the NABD-CP fusion protein while bound to the NAM; (b) contacting the NAM:NABD-CP complex with a target molecule (TM), wherein binding of the NAM:NABD-CP complex to the TM results in formation of a NAM:NABD-CP:TM complex; and (c) isolating the NAM of said
  • Also provided are methods for isolating a nucleic acid sequence encoding a polypeptide that binds to a target molecule comprising the steps of: (a) providing a first NAM:NABD-CP complex comprising: (i) a first NAM comprising a TS of a first NABD and further comprising a sequence encoding a first NABD-CP fusion protein, wherein the first NABD is capable of binding to the TS of the first NABD; and (ii) the first NABD-CP fusion protein while bound to the first NAM; (b) providing a second NAM:NABD-CP complex comprising: (i) a second NAM comprising a TS of a second NABD and further comprising a sequence encoding a second NABD-CP fusion protein, wherein the second NABD is capable of binding to the TS of the second NABD; and (ii) the second NABD-CP fusion protein while bound to the second NAM; (c) contacting the first NAM:NABD
  • a candidate polypeptide is produced from a nucleic acid molecule (NAM) that encodes the CP together with a nucleic acid binding protein (NABD) as a fusion protein (NABD-CP fusion protein).
  • NAM nucleic acid molecule
  • NABD nucleic acid binding protein
  • the NAM encoding the NABD-CP fusion protein also contains a target sequence (TS) to which the NABD can bind so that a complex containing the NABD-CP fusion protein and the NAM can be formed (NAM:NABD-CP complex).
  • TS target sequence
  • NAM:NABD-CP complex a complex containing the NABD-CP fusion protein and the NAM can be formed
  • This complex can serve both to exhibit the binding characteristics of the CP and to include the nucleotide sequence encoding the CP.
  • such complexes can be conveniently used for screening and isolating CPs with binding properties of interest and at the same time isolating and recovering the nucleotide sequence encoding such CPs.
  • FIG. 1 A specific example of the DNA Display method is provided in Figure 1, where the NABD is a Zn finger protein (ZF), the CP is a Fab, and the NAM is an Escherichia coli vector containing a Zn finger protein target sequence (ZF-TS) and encoding the Zn finger and Fab as a fusion protein (ZF/Fab).
  • ZF-TS Zn finger protein target sequence
  • ZF/Fab fusion protein
  • an NAM containing the ZF-TS and encoding the ZF/Fab is introduced into E. coli cells, and the cells are placed under conditions in which plasmid replication and ZF/Fab translation occur (Figure 1, upper left).
  • the ZF/Fab binds to the ZF-TS within the cell to form a NAM:ZF/Fab complex ( Figure 1, upper right).
  • NAM:ZF/Fab complex-containing lysate is used in screening methods to determine whether or not the Fab binds to the target antigen of choice.
  • the target antigen is attached to a bead, such as a magnetic bead.
  • NAM:ZF/Fab complex-containing lysate is then mixed and incubated with the magnetic bead-bound antigens ( Figure 1, lower right), and any NAM:ZF/Fab complex-containing lysate that bound to the magnetic bead-bound antigens can be separated from the remainder of the lysate by washing the beads and removing the wash from the beads ( Figure 1, lower left).
  • the NAM of the antigen-bound NAM:ZF/Fab complex can then be isolated and used in further experimental methods and/or can be at least partially sequenced to elucidate some or all of the amino acid sequence of the Fab that bound to the antigen.
  • the nucleic acid molecule (NAM) used in the methods provided herein encodes the candidate polypeptide (CP) and a nucleic acid binding protein (NABD), and also contains a target sequence (TS) to which the NABD protein can bind.
  • CP candidate polypeptide
  • NABD nucleic acid binding protein
  • TS target sequence
  • the NAM will typically contain one or more additional components as appropriate according to the desired method for obtaining a desired number of NAM copies and for obtaining a desired number of translated CP copies.
  • a cell system can be used for NAM amplification and CP expression, and the components of the NAM will typically include those components of a vector conducive to vector amplification and gene expression in the cell.
  • Such components can include, but are not limited to, a selectable marker or selection gene that permits selection of cells that have been transformed with the NAM, a replication sequence that permits the plasmid to be replicated in the cell, transcriptional regulatory sequence(s) that control transcription of the CP and typically permit inducible expression of the CP transcript.
  • host cells are transformed with the vectors.
  • the successful transformants are typically selected by growth in a selective medium or under selective conditions, e.g., an appropriate antibiotic such as ampicillin. This selection can be done on solid or in liquid growth medium.
  • the cells are grown at a high density (e.g., 10 8 to 10 9 transformants per m 2 ) on a large surface of, for example, L-agar containing the selective antibiotic to form essentially a confluent lawn.
  • L-broth with antibiotic selection
  • Growth in liquid culture can be more convenient to yield larger sized libraries.
  • the ratio of CP copies to NAM copies can be controlled so that the NAMs are saturated with CPs, without a vast excess of CPs. Too few CPs could result in NAMs with free binding sites that might be filled by different CPs from other cells whose lysates are admixed prior to the target molecule-binding selection process, thus breaking the connection between the genetic information and the CP. Too many CP copies could lead to unduly high occupancy of available target molecule during the target-molecule-binding selection process.
  • any of a variety of known origin-of-replication sequences to control vector number and/or any of a variety of known inducible promoters, such as any of the promoters selected from the group consisting of the araB, lambda pL, (which can be induced by nalidixic acid or heat or both), trp, lac, T7, T3, and tac or trc (these latter two are trp/lac hybrids) promoters to control fusion protein number.
  • a regulated promoter is also useful to limit the amount of time that the CPs are exposed to cellular proteases. By inducing the promoter a short time before lysing the cells containing a library, one can minimize the time during which proteases act.
  • Variation of NAM and CP copy numbers for improved results can be performed using routine methods known in the art.
  • a vector such as a plasmid
  • non-inducing conditions to minimize exposure of the CP to cellular proteases and to minimize exposure of the cell to the possibly deleterious effects of the CP
  • partial induction can be achieved with as little as about 10 "5 % of L-arabinose.
  • An exemplary manner to achieve partial induction can include growing the cells in 0.1% glucose until about 30 min. before the cells are harvested; then, 0.2 to 0.5% L-arabinose is added to the culture to induce expression of the fusion protein.
  • Other methods to express the protein controllably are available.
  • the NAM need not contain elements such as an origin of replication or a selectable marker.
  • the NAM can be produced by in vitro amplification methods such as PCR; such a NAM would not require an origin of replication or a selectable marker, and would only require that the NAM be capable of being amplified according to the desired in vitro amplification method to be implemented.
  • the CP can be produced by in vitro translation or linked or coupled in vitro transcription:translation.
  • the components of the NAM are selected according to the requirements of the particular translation or transcription:translation system used, for example, a T7 polymerase promoter is included in an NAM used for in vitro transcription:translation in some reticulocyte lysate systems.
  • the NAMs are constructed so that the CP is expressed as a fusion product; the CP is fused to a nucleic acid binding domain (NABD).
  • NABD nucleic acid binding domain
  • a NABD used in the present methods typically exhibits high avidity binding to a particular nucleotide sequence or tertiary structure and a tolerance for expression as a fusion product such that its nucleic acid binding properties are not significantly adversely affected.
  • the half-life of a NAM:NABD complex produced in the present method is typically long enough to allow screening to occur. For example, the half-life can be at least 15 min and often between one to four hours or longer. Data on DNA half-lives are available for numerous DNA-binding proteins.
  • the arc repressor of phage P22 has a dissociation half-life of 80 min (see, e.g., Knight et al., J. Biol. Chem. 264, 3639-3642 (1989), Vershon et al., J. MoI. Biol. 195, 323-331 (1987)).
  • dissociation half-life can be determined by standard biochemical procedures (see, e.g., Bourgeois, Methods Enzymol. 21D, 491-500 (1971) (filter binding assay), Knight & Sauer, J. Biol. Chem. 264, 13706-13710 (1989) (DNA modification protection assay)).
  • the high stability of the Zn-finger:DNA complex can be particularly advantageous for purposes of the present methods: for the binding affinity selection or NAM- isolating step to succeed, the CP and the NAM that encodes the CP should not dissociate so that isolation of the CP does not also lead to isolation of the NAM encoding the CP. In fact, in some cases a longer half-life may be preferred.
  • Suitable NABDs that can be used in the present methods include proteins or fragments thereof selected from a large group of known DNA-binding proteins, including transcriptional regulators and proteins that serve structural functions on DNA. Examples include: proteins that recognize DNA by virtue of a helix-turn-helix motif, such as the phage 434 repressor, the lambda phage cl and cro repressors, and the E.
  • coli CAP protein from bacteria and proteins from eukaryotic cells that contain a homeobox helix-turn-helix motif; proteins containing the helix-loop-helix structure, such as myc and related proteins; proteins with leucine zippers and DNA binding basic domains such as fos and jun; proteins with TOlT domains such as the Drosophila paired protein; proteins with domains whose structures depend on metal ion chelation such as the Zn finger domain of SEQ ID NO:2, as encoded by SEQ ID NO: 1, the CyS 2 Hi S 2 zinc fingers found in TFIIIA, Zn 2 (CyS) 6 clusters such as those found in yeast GaI 4 , the CyS 3 HiS box found in retroviral nucleocapsid proteins, and the Zn 2 (CyS) 8 clusters found in nuclear hormone receptor-type proteins; the phage P22 Arc and Mnt repressors (see Knight et al., J.
  • proteins could be used that bind to the NAM indirectly, by virtue of binding another protein bound to the NAM. Examples of these include yeast Gal80 and adenovirus ElA protein. Phage coat proteins, which associate with DNA by encapsidation of the DNA in a phage coat, are typically not employed in the present methods.
  • the NABD is a Zn finger binding domain (ZF), such as the Zinc finger binding domain of SEQ ID NO:2.
  • Zn finger binding domains have a high "on rate" kinetic binding constant for binding the target nucleotide sequence, resulting in a large half-life of dissociation of Zn fingers from the bound nucleic acid molecule.
  • Zn finger binding domains with particularly favorable binding properties is the Zn finger binding domain of SEQ ID NO:2, which binds the target sequence GGGGCTGGGGGCGGTGTCT (SEQ ID NO: 5).
  • Zn finger binding domains can be engineered to modify their sequence specificity, as know in the art exemplified in Segal et al., J MoI Biol. 2006 Aug 11; [Epub ahead of print], and references cited therein, all of which are incorporated by reference herein in their entirety.
  • the Zn finger binding domain of SEQ ID NO:2 can be modified according to known methods and principles to modify their sequence specificity.
  • a candidate polypeptide (CP) used in the present methods can be any polypeptide for which the binding ability to a target molecule can be determined.
  • a CP can be a full-length protein or a fragment thereof, or a sequence variant thereof.
  • a CP can vary in size from a small peptide to a full-length protein, and can include a protein assembly (e.g., a homo- or hetero- dimer, trimer or other multimer) such as an antibody or other naturally occurring protein assembly.
  • the CP typically contains at least or at least about 6, 7, 8, 9, 10, 12, 15, 18, 20, 25, 30, 35, 40, 45 or 50 amino acids.
  • the CP can be expressed alone or as a portion of a fusion protein (e.g., a fusion protein with an NABD).
  • one of the polypeptides in the assembly can be expressed as a portion of a fusion protein with the NABD while the remaining polypeptides of the assembly are not expressed in fusion with an NABD.
  • two or more of the polypeptides in the assembly can be expressed as a portion of a fusion protein with the NABD while any remaining polypeptides of the assembly, if present, are not expressed in fusion with an NABD.
  • a CP used in the present methods is not particularly limited.
  • a CP can be a naturally occurring protein, or can be a variant of a naturally occurring protein, such as a mutant form or a truncation/fragment of the naturally occurring protein or mutant thereof.
  • a CP also can be a designed protein or peptide not based on any particular naturally occurring amino acid sequence.
  • Exemplary CPs include, but are not limited to, cell surface proteins, growth factors, peptide hormones, enzymes, cellular adhesion proteins, or antibodies, or fragments of any of these.
  • the nucleotide sequence encoding the CP is not particularly limited: the sequence can be obtained from a natural source, can be totally synthetic, or can be a nucleotide sequence that has been manipulated by any of a variety of known methods.
  • the CP-encoding nucleotide sequence is derived from a cDNA library; in another example, the CP-encoding nucleotide sequence is derived from an animal tissue source (e.g., spleen); in another example, the CP-encoding nucleotide sequence is, at least in part, modified to contain a randomized sequence; in another embodiment.
  • a CP can contain one or more post-translational modifications, such as those found in naturally occurring peptides and proteins, which can provide additional diversity to the CPs, can increase the folding efficiency, stability, solubility or other desired property of the CP.
  • the CP can contain one or more amino acid residues involved in phosphorylation, glycosylation, sulfation, isoprenylation (or other lipidylation), enzymatic cleavage, or other post-translational modification known in the art.
  • the post- translational modification site can be simply a single residue (e.g., serine for phosphorylation) or a complex consensus sequence, as desired.
  • post-translational modification can vary according to, inter alia, the type of cell in which the CP is expressed (e.g., bacterial, insect, mammalian).
  • post-translational modifications can be selected/controlled according to the chosen expression system.
  • An exemplary CP is an antibody or antibody fragment that retains the specific binding ability of the antibody.
  • the antibody can be a member of any immunoglobulin class, including IgG, IgM, IgA, IgD and IgE.
  • antibody fragments include, but are not limited to, Fab, Fab', hsFv, F(ab) 2 , single-chain Fvs (scFv), small immune proteins, Fv, dsFv diabody and Fd fragments.
  • the fragment can include multiple chains linked together, such as by disulfide bridges.
  • An antibody fragment generally contains at least about 50 amino acids and typically at least about 200 amino acids, or at least 50 amino acids and typically at least 200 amino acids.
  • the CP is a Fab fragment, which is an antigen-binding antibody fragment containing a Fab heavy chain which contains one variable heavy domain (V H ) and one constant heavy domain 1 (C HI ), and a Fab light chain which contains one variable light (V L ) domain and one constant light (C L ) domain.
  • each of the different CPs can be a different antibody portion, which can assemble with the other CPs.
  • a two different CPs can assemble to form a Fab, where a first CP is a Fab heavy chain, and a second CP is a Fab light chain.
  • the antibody or antibody fragment can be from any of a variety of sources, such as mammalian (e.g., rabbit, mouse, primate or human).
  • the antibody or antibody fragment can be humanized, so that administration to a human does not provoke an immune response.
  • the antibody or antibody fragment can be an autoantibody.
  • libraries of two or more different CPs each complexed with their respective encoding NAM. These libraries can be contacted with the target molecule according to the methods provided herein and any member of the library that demonstrates the desired affinity to the target molecule can be isolated. Thus, the presently provided methods can be used to screen polypeptide-containing libraries for the ability to bind a target molecule.
  • the libraries of different CPs can be derived from any collection of nucleotide sequences encoding polypeptides.
  • the library can be derived from a cDNA library, a synthetic library, or any other polypeptide-encoding nucleotide library known in the art.
  • the library can be selected to contain nucleotides encoding particular types of proteins, such as membrane proteins, antibodies, related enzymes, proteins having a particular domain in common, peptide hormones, and the like.
  • the library can be a library of polynucleotides encoding antibodies or antibody fragments.
  • the number of different CPs of a library provided herein can be two or more, but typically can be at least, or at least about 10, 20, 30, 40, 50, 60, 70, 80, 100, 150, 200, 300, 400, 500, 750, 1000, 2000, 5000, 10,000, 50,000, 10 5 , 10 6 , 10 7 , 10 8 , or can fall within a range defined by any two of these values.
  • CPs immediately adjacent to other polypeptide sequences such as a NADB
  • fusion proteins containing CPs separated from other portions of the fusion protein by spacer residues.
  • the CPs can be separated by spacers that allow the CPs more flexibility in accessing and binding the TM.
  • spacers typically permit separation of different domains of a fusion protein.
  • the distance between domains or regions or regions of the fusion protein can be as little as one residue or as many as five to ten to up to about 100 residues.
  • CPs can be separated from other fusion protein regions by a spacer containing 20 to 30 amino acids.
  • the number of spacer residues, when present, can be at least two to three or more but usually will be less than eight to ten.
  • the spacer residues can be somewhat flexible, for example comprising oligoglycine, to provide the CPs with the ability to interact with sites in a large TM binding site relatively unconstrained by attachment to the NABD or other domain of the fusion protein.
  • Rigid spacers such as, e.g., oligoproline, also can be inserted separately or in combination with other spacers, including glycine residues.
  • a spacer also can serve to orient one domain of the fusion protein with respect to another, such as by employing a turn between the two sequences, as might be provided by a spacer of the sequence Gly-Pro-Gly, for example.
  • a spacer of the sequence Gly-Pro-Gly, for example.
  • Cys residues it may be desirable or necessary to add Cys residues at either or both ends of each domain being oriented. The Cys residues would then form disulfide bridges to hold the domains together in a loop.
  • the CP or a portion thereof is typically expressed in fusion with the NABD.
  • the NABD-CP fusion protein can be provided by the experimenter or by expression in vitro or in vivo.
  • the particular system for NABD-CP fusion protein expression can be selected, if desired, according to any preferred expression level, post-translational modification, cell compartmentalization, ease or expense, or other desired factor relevant to production of the NABD-CP fusion protein.
  • In vivo systems for expression of the NABD-CP fusion protein is not particularly limited, but typically will be a well-established protein expression system that is selected according to the preferred form of the NABD-CP fusion protein product.
  • Exemplary systems include, but are not limited to, Escherichia coli, Saccharomyces cerevisiae, Spodoptera frugiperda larvae ovarian-derived cells, green African monkey kidney epithelium-derived cells, and other known systems.
  • the NABD portion of the NABD-CP fusion protein can bind the NAM that encodes the NABD-CP fusion protein to form a NAM:NABD-CP complex.
  • the NAM is contacted with the NABD-CP fusion protein under conditions suitable for NAM:NABD-CP complex formation.
  • the NAM:NABD-CP complex can form under physiological conditions within a cell, or under the conditions of in vitro translation.
  • NABD-CP complex examples include PBS (137 mM NaCl, 2.7 mM KCl, 10 mM phosphate buffer pH 7.4), or sonication buffer (0.5 mM DTT, 0.05% Tween 20, 0.1 mg/ml ssDNA (Salmon sperm DNA), 10 mg/ml BSA (or 5% milk), 50 mM Na-Glutamate, 100 ⁇ M ZnC12, 10 mM HEPES, pH 7.4).
  • PBS 137 mM NaCl, 2.7 mM KCl, 10 mM phosphate buffer pH 7.4
  • sonication buffer 0.5 mM DTT, 0.05% Tween 20, 0.1 mg/ml ssDNA (Salmon sperm DNA), 10 mg/ml BSA (or 5% milk), 50 mM Na-Glutamate, 100 ⁇ M ZnC12, 10 mM HEPES, pH 7.4
  • the NAM:NABD-CP complex is then harvested, if necessary, to obtain the NAM:NABD-CP complex in a form suitable for being contacted with a target molecule.
  • the NAM:NABD-CP complex is formed under in vitro translation conditions, no additional steps are required, but further isolation steps can be performed, if desired.
  • the NAM:NABD-CP complex is formed in vivo, the cells are typically lysed in harvesting the NAM:NABD-CP complex.
  • Cell lysis can be performed using to any known method, according to the type of cell used. For example, cell lysis can be performed using sonication, glass beads, chemical lysis or other known method.
  • NAM:NABD-CP complex-containing cell lysate without further purification, can typically be used in steps of contacting the NAM:NABD-CP complex with the target molecule, but further isolation steps can be performed, if desired.
  • each NAM:NABD-CP complex is typically formed prior to mixing the different NAM:NABD-CP complexes.
  • TM target molecule
  • a target molecule (TM) used in these methods include, by way of example and not limitation, cell surface molecules, growth factors, hormones, enzyme substrates, interferons, interleukins, intracellular and intercellular messengers, natural product small molecules, drugs, lectins, cellular adhesion molecules, antigens, such as a bacterial surface molecule or a viral surface molecule, and the like.
  • TMs can be peptides, proteins, nucleic acids, carbohydrates, lipids, or other organic compounds, or metals, or other inorganic compounds.
  • the TM used in the methods provided herein can be bound by a polypeptide, such as a candidate polypeptide used in the presently provided screening methods.
  • the TM also can be either detected or isolated when bound by a candidate polypeptide.
  • the TM can, when bound to the CP, be detected and/or isolated so that the NAM encoding the bound CP can be isolated.
  • the TM itself can be detectable or isolatable, or can be attached to a detectable or isolatable moiety, or a moiety that can bind to a detectable or isolatable moiety.
  • the TM can be a protein containing a C- terminal His tag, where the NAM:NABD-CP-bound TM can be isolated using a Ni- containing support or bead.
  • TM can be a protein such as lysozyme, and the NAM:NABD-CP-bound lysozyme can be isolated by immunoprecipitation using polyclonal anti-lysozyme antibodies.
  • Detectable moieties can be used for isolating the CP-encoding NAMs in methods such as flow cytometry, where components of a mixture are separated according to the presence or absence of a particular signal.
  • Isolatable moieties can be used to facilitate isolation of any NAM:NABD-CP-bound TM.
  • the attachment of a TM to a moiety can be covalent or non-covalent, and is typically of sufficiently high affinity as to not result in detachment of the TM and moiety during steps of isolating the NAM:NABD-CP-bound-TM from unbound NAM:NABD-CP.
  • Any detectable or isolatable moiety known in the art can be used, according to the desired properties of the moiety and any requirements of the TM or the NAM:NABD-CP-bound TM complex.
  • Exemplary detectable moieties include, but are not limited to fluorophores, chromophores, quantum dots, radionuclides, and the like.
  • Exemplary isolatable moieties can be a molecule or composition, and can include substrates and structures such as matrices, supports, beads, plates and arrays.
  • Materials that can be used for an isolatable moiety include any material that can be used as affinity matrices, supports or beads for chemical and biological molecule syntheses and analyses, such as, but are not limited to: organic or inorganic polymers, biopolymers, natural and synthetic polymers, including, but not limited to, agarose, cellulose, nitrocellulose, cellulose acetate, other cellulose derivatives, dextran, dextran-derivatives and dextran co-polymers, other polysaccharides, gelatin, polyvinyl pyrrolidone, rayon, nylon, polyethylene, polypropylene, polybutylene, polycarbonate, polyesters, polyamides, vinyl polymers, polyvinylalcohols, polyvinylidenedifluoride (PVDF), polystyrene and polystyrene copolymers, polystyrene cross-linked with divinylbenzene or the like, acrylic resins, acrylates and acrylic acids, acrylamides, polyacryl
  • CPG controlled-pore glass
  • silica gels silica gels, ceramics, paper, natural sponges, insoluble protein, surfactants, red blood cells, metals (including metal ions; e.g., steel, gold, silver, aluminum and copper), metalloids, magnetic materials (including TeflonTM coated magnetic materials and magnetic beads), Wang resin, Merrifield resin, SephadexTM, SepharoseTM, nylon, dextran, chitin, sand, pumice, dendrimers, buckyballs, or other commercially available medium.
  • CPG controlled-pore glass
  • Exemplary supports include, but are not limited to flat supports such as glass fiber filters, silicon surfaces, glass surfaces, latex beads, magnetic beads, nitrocellulose membranes, tissue culture plates, microarrays, metal surfaces (steel, gold, silver, aluminum and copper) and plastic materials.
  • Exemplary molecules include biotin and flag polypeptide. Attachment of the TM to the detectable or isolatable moiety can be performed in accordance with the particular properties of the TM and the moiety, using methods known in the art. Contacting the CP and TM
  • the methods provided herein include a step of contacting the complex containing the CP and the NAM with the TM under conditions suitable for binding between the CP and the TM, but generally unsuitable for non-specific association of the CP and TM.
  • the contacting can be performed by any of a variety of know methods, such as mixing of a CP-containing liquid with a TM-containing liquid, flowing or mixing of a CP-containing liquid with a column or solid support containing a TM, and other analyte contacting methods known in the art.
  • Non-specific association of the CP to the TM can typically be reduced or eliminated using known buffer conditions containing compounds that block and/or reduce non-specific association, such as inclusion of a random or negative control molecule such as BSA or milk protein, a detergent or denaturant such as a non-ionic detergent or urea, a salt to increase the ionic strength of the contacting conditions, and other compounds known to serve such roles.
  • a random or negative control molecule such as BSA or milk protein
  • a detergent or denaturant such as a non-ionic detergent or urea
  • salt to increase the ionic strength of the contacting conditions
  • Exemplary conditions for conditions suitable for binding between the CP and the TM, and that inhibit or reduce non-specific association of the CP and TM include: 0.5 mM DTT, 0.05% Tween 20, 10 mg/ml BSA, 50 mM Na-Glutamate, 100 ⁇ M ZnCl 2 , 10 mM HEPES, pH 7.4; or PBS plus 0.1% Tween 20 with 5% dry milk powder.
  • the binding between the CP and the TM is characterized by a K d in the range of 10 "2 to 10 "15 mole/L, generally, 10 "6 to 10 "15 , 10 “7 to 10 “15 and typically 10 "8 to 10 "15 mole/L (and/or a K a of 10 5 -10 12 , 10 7 -10 12 , 10 8 -10 12 L/mole).
  • a CP that binds to the TM with the desired level of specificity typically binds to the TM with at least, or at least about 2-fold and typically at least, or at least about 5-fold, 10-fold, 50-fold, 100-fold, or more, greater affinity (K a or K eq ) than for another molecule (e.g., a random or negative control molecule such as lysozyme, BSA or milk protein).
  • Typical conditions for detecting and determining binding affinity constants or for determining the specificity of binding include physiological conditions, such as PBS (137 mM NaCl, 2.7 mM KCl, 10 mM phosphate buffer pH 7.4).
  • nonspecific association of a CP and a TM is typically at least, or at least about 2-fold and typically at least, or at least about 5-fold, 10-fold, 50-fold, 100-fold, weaker than the specific binding of the CP to the TM.
  • any CP that binds the TM will form a CP: TM complex.
  • the CP will typically be expressed as a fusion protein with a NABD and will be in complex with the NAM encoding the CP.
  • binding of a CP to a TM typically results in formation of a NAM:NABD-CP:TM complex.
  • the complex formed between the CP and TM can then be subjected to one or more isolation steps to obtain the NAM encoding the CP that bound to the TM. Isolating the NAM
  • the complex is subjected to one or more separation steps that result in isolation of the NAM encoding the CP that bound the TM. Any of a variety of separation steps can be performed and any of a variety of combinations of two or more separation steps can be performed as appropriate to yield the NAM at a desired level of purity.
  • the separation steps to be performed can be determined according to the TM used, and/or according to the desired final state of the NAM to be isolated.
  • Separation steps typically include one or more steps of separating the CP (and encoding NAM) that bound the TM from the CP (and encoding NAM) not bound to the TM, and one or more steps of harvesting the NAM from the CP: TM complex that contains the NAM encoding the bound CP.
  • Methods for separating a TM-bound CP are typically performed by separating all or a selected subset of TMs from the non-bound CPs by any known method, which typically is a method compatible with the TM used in the screening method.
  • a TM that is attached to an isolatable moiety such as a magnetic bead is separated from non-bound CPs by washing the magnetic beads with an appropriate wash solution and removing the wash solution from the magnetic beads using, e.g., a magnetic plate to hold the magnetic beads fixed while the wash solution is removed.
  • the TM can be attached to a solid support and the CP-containing solution can be flowed over/through the solid support.
  • separation of the TM from the non-bound CP can be performed simply by allowing the CP-containing solution to wash flow/through the solid support so that the solution is no longer in contact with the solid support.
  • the step of contacting a CP and TM and the step of isolating the NAM in the present methods can be performed in a single step of flowing the CP-containing solution over a solid support, where the isolating component of the wash step is achieved when the CP-containing solution is no longer in contact with the TM-bound support.
  • the present methods do not require a temporal separation between the contacting and isolating steps — the two can be performed as a continuous process as occurs when a solution is flowed over a solid support — instead, the inclusion of both a contacting and separation step in the present methods merely refers to a process in which CP and TM can be brought together and NAM encoding CP not bound to TM can be separated from NAM encoding CP bound to TM.
  • the degree and stringency of washing required can readily be determined for each CP/TM of interest. Control can be exerted over the binding characteristics of the peptides recovered by adjusting the conditions of the binding incubation and the subsequent washing. For example, the temperature, pH, ionic strength, divalent cation concentration, and the volume and duration of the washing will select for CPs within particular ranges of affinity for the receptor. In one example, selection based on slow dissociation rate, which is usually predictive of high affinity, is performed. This can be accomplished either by continued incubation in the presence of a saturating amount of free TS, or by increasing the volume, number, and length of the washes.
  • NAM is typically harvested in such a manner that the NAM can be further used and/or analyzed in assessing, confirming, or refining the results.
  • the CP: TM complex can be separated using a buffer solution such as a high salt solution or a protein-denaturing solution, so that the NAM is no longer present in complex with the TM. If desired, the TM can be reused or discarded.
  • Additional NAM harvesting methods that can be performed include any of a variety of known nucleic acid isolation procedures, such as mini preps or other known methods.
  • NAM harvesting methods can include maintaining the CP and NAM in the same complex, for example, when separating the NAM from the TM, it is not required to do so, provided that the NAM is maintained in a form that permits its further use and/or analysis.
  • the isolating step comprises immunoprecipitating the NAM:NABD-CP:TM complex.
  • a solid support can have attached thereto antibodies (monoclonal or polyclonal) that recognize the CP-TM complex or simply the TM.
  • CP-TM complex or TM is bound by such immobilized antibodies
  • the TM-bound CPs and, hence, the associated NAM
  • the NAM bound to the solid support can then be isolated. Any of a variety of immunoprecipitation methods known in the art can be performed in such a NAM isolation step.
  • the above steps of combining NABD-CP with TM to form a NAM:NABD-CP complex, contacting the NAM:NABD-CP complex with the TM and isolating any NAM encoding a CP that bound to the TM can be repeated one or more times, as desired, in order to further validate a putative hit, to enrich the sample with actual hits, or to increase the threshold degree of binding in order to select CPs that with the highest binding affinities.
  • the number of additional cycles that can be performed are not limited and can be selected according to the efficiency of each cycle and the desired number and quality of the resulting hits. Exemplary numbers of additional cycles are at least one, at least two, at least three, at least four, at least five, or more additional cycles.
  • the contacting and/or isolating conditions and steps can be varied as desired (e.g., increasingly higher salt conditions in the contacting step) and the TM can be attached to the same or different detectable or isolatable moiety.
  • screening steps complementary to the above steps can be performed in validating hits or increasing the threshold selection of CPs.
  • CPs encoded by the NAMs isolated in the above steps can be individually assayed and those displaying the most desired properties can be identified as the preferred CPs.
  • individually assaying CPs NAMs isolated in the above steps can be transfected into cells to form colonies, and the colonies can be grown to each produce a single NAM:NABD-CP complex, and the complexes can be harvested in a manner similar to that provided above. Each NAM:NABD-CP complex can then be separately contacted with a TM and assayed for its binding affinity.
  • an ELISA assay can be performed using an antibody to the NAM, NABD or non-varied portion of the CP.
  • the wells having the highest ELISA values can be identified as containing CPs with the most desired binding properties. Any of a variety of similar methods can be implemented as known in the art, to provide measurement methods for distinguishing different CPs identified in the contacting and isolating steps provide above.
  • a CP can be contacted with a TM and with one or more negative controls under assay conditions that permit quantitative estimate of the binding affinity of the CP to the TM and controls (see, e.g., Figure 4). Comparison of the binding affinity of CP for TM versus CP for the controls can demonstrate the degree of selectivity of CP for TM.
  • a CP can be serially diluted in quantitative assays, or the contacting conditions can be varied in stringency (e.g., increased in salt concentration) in quantitative assays, and the avidity (e.g., binding constant) of the CP for the TM can be estimated.
  • a CP can be contacted with a TM bound to an isolatable moiety (e.g., TM on a 96-well plate), and saturating concentrations of TM not bound to an isolatable moiety can be added and time points can be monitored to measure the off-rate of the CP:TM complex.
  • a CP can be contacted with a TM bound to an isolatable moiety (e.g., TM on a 96-well plate), and saturating concentrations of different fragments of TM not bound to an isolatable moiety can be added in different reaction vessels (e.g., wells), and those reaction vessels in which the CP:TM complex is decreased or not detectable can indicate that the added TM fragment contains a site at which the CP bound to the TM, while those reaction vessels in which the CP:TM complex is essentially unchanged can indicate that the added TM fragment does not contain a site at which the CP bound to the TM.
  • Isolated NAM TM bound to an isolatable moiety
  • the above steps result in an isolated NAM encoding and CP that bound to the TM.
  • the isolated NAM can be used in further experimental procedures, and/or its nucleotide sequence can be analyzed. Further experimental procedures that can be performed include, but are not limited to, further refinement and analysis of the CP according to methods such as those provided above, modification of the CP-encoding nucleotide sequence to create one or more second-generation CPs that can be further screened or analyzed, testing the CP for affinity to TMs that are similar to or dissimilar to the TM used in the initial screen.
  • nucleotide sequence modification methods can be used, including but not limited to, cassette mutagenesis, error-prone PCR, recombination of sequences from NAMs encoding other CPs (e.g., other hits identified in the screen), and other methods known in the art.
  • Particular methods for modifying and evolving the CP-encoding nucleotide sequence include Gene Site Saturation Mutagenesis (GSSM) as described in U.S. Patent No. 6,171,820, No. 6,562,594, and No. 6,764,835, and Synthetic Ligation Reassembly (SLR) as described in U.S. Patent No. 6,537,776 and No. 6,605,449, each of which is incorporated herein.
  • GSSM Gene Site Saturation Mutagenesis
  • SLR Synthetic Ligation Reassembly
  • the resultant CPs/NAMs can be screened according to the methods provided herein or otherwise known in the art.
  • At least a portion of the CP-encoding nucleotide sequence of the NAM can be determined. While it is possible for one skilled in the art to elucidate the entire nucleotide sequence of an isolated NAM, it may be only necessary or desirable to elucidate a portion thereof, which typically is the portion considered to encode a varying region of the CP that can contribute to the TM-binding ability of the CP.
  • nucleotide sequence of the entire NAM need not be elucidated, nor need the entire sequence of the antibody or fragment thereof necessarily be elucidated; instead sequences of only the variable domain(s) can provide substantial information regarding the amino acids of the antibody or antibody fragment that can contribute to the TM-binding ability of the antibody or antibody fragment. If desired for completeness, the nucleotide sequences of the constant domains also can be elucidated.
  • the portion of the CP-encoding nucleotide sequence of the NAM to be determined can be selected by one skilled in the art according to the CP used and according to the desired completeness of information about the CP. Assembly DNA Display
  • the methods provided in the present application can be used to increase the complexity of polypeptide libraries by combining a plurality of libraries of polypeptides which can associate with each other to form a complex, where these complexes can bind to a target molecule.
  • the methods provided above can be further expanded to generate and screen combinatorial libraries of different CPs that associate with one another and that can contribute to binding of a TM.
  • a first library of recombinant antibody light chains and a second library of recombinant antibody heavy chains can be associated to both make a much larger combinatorial library of novel antibodies.
  • Assembly DNA Display is useful for generating libraries of novel antibodies from heavy and light chain libraries, screening the antibodies for desired binding properties, and recovering nucleic acid sequences encoding the heavy and light chains in the most promising antibodies.
  • Methods for combining libraries in isolating a nucleic acid sequence encoding a polypeptide that binds to a target molecule can comprise the steps of: (a) providing a first NAM:NABD-CP complex comprising: (i) a first NAM comprising a TS of a first NABD and further comprising a sequence encoding a first NABD-CP fusion protein, wherein the first NABD is capable of binding to the TS of the first NABD; and (ii) the first NABD-CP fusion protein while bound to the first NAM; (b) providing a second NAM:NABD-CP complex comprising: (i) a second NAM comprising a TS of a second NABD and further comprising a sequence encoding a second NABD-CP fusion protein, wherein the second NABD is capable of binding to the TS of the second NABD; and (ii) the second NABD-CP fusion protein while bound to the second NAM; (c) contacting the steps of: (
  • first and second NAM:NABD-CP complexes associate with one another, and in some embodiments this association is in the absence of TM, while in other embodiments this association is only in the presence of a TM or other ligand.
  • Exemplary first and second NAM:NABD-CP complexes that associate with one another include a first NAM:NABD-CP complex in which the first CP is a Fab light chain, and a second NAM:NABD-CP complex in which the second CP is a Fab heavy chain.
  • each NAM:NABD-CP complex can be formed as provided herein above, and each TM can be detectable or isolatable as described above. Further, the contacting and isolating steps are performed under similar conditions, with variations as explained below to address the association of the multiple NAM:NABD-CP complexes. Accordingly, any particular detail regarding the molecule used or the method performed that is not explicitly described in the present section can have the characteristics of those described above, as understood by one skilled in the art.
  • the present method is not limited to only use of a first and second NAM:NABD-CP complex, and formation of a (NAM:NABD-CP) 2 :TM complex refers to a complex between two or more NAM:NABD-CPs and a TM, and is not limited to instances in which only two NAM:NABD-CPs are complexed with a TM.
  • a first and second NAM:NABD-CP complex are provided.
  • each NAM:NABD-CP complex is formed in a separate system, such as a separate cell or a separate in vitro system, and do not contact one another until after formation of each NAM:NABD-CP complex.
  • the NABD and the TS of the NABD can be the same or different, and can be selected according to the preferences of one skilled in the art.
  • the affinity of each NABD to the TS is sufficiently high so that the first and second NABDs substantially do not dissociate so that the CP is no longer in complex with its encoding NAM.
  • At least two different NAM:NABD-CP complexes are formed in the same system, such as the same cell or the same in vitro system.
  • the TS of the first NABD differs from the TS of the second NABD so that each NAM:NABD-CP complex that forms retains the CP bound to its respective encoding NAM.
  • NABDs that bind to different TSs are known in the art, and any of a variety can be used in combination.
  • the NABDs can be different Zn finger domains that recognize different target sequences.
  • the selection sequences can be the same or different, but are typically different in systems in which both a first and second NAM:NABD-CP complex are formed, thus ensuring introduction of both the first and the second NAM into the system (e.g., cell).
  • the first and second NAM:NABD-CP complexes associate with each other.
  • these complexes can associate in the absence of TM, while in some embodiments, a ligand, such as a TM is required for the first and second NAM:NABD-CP complexes to associate.
  • the association of the first and second NAM:NABD-CP complexes can be due to any intramolecular interaction between the components of the first and second NAM:NABD-CP complexes.
  • association will be via multimerization (e.g., dimerization) of the first and second NABDs or multimerization (e.g., dimerization) of the first and second CPs.
  • first and second NAM:NABD-CP complexes that associate via the first and second CPs a first CP can be a Fab light chain and a second CP can be a Fab heavy chain, where the association between the Fab light chain and Fab heavy chain includes contacts between substantially unchanged amino acid sequences of each chain.
  • the type of multimer that can be formed is not limited, and can be a dimer, a trimer, a tetramer, a pentamer, a hexamer, or larger multimer.
  • the number of different CPs can be two or more, up to as many different CPs as components of the multimer.
  • both CPs of the first and second NAM:NABD-CP complexes bind to the TM.
  • the combination of CPs can be screened for their ability to bind the TM.
  • each CP can be separately screened for its contribution to the binding to the TM.
  • a single first CP can be used and screened in conjunction with two or more second CPs for their ability, when complexed, to bind the TM.
  • two or more first CPs can be combined combinatorially with two or more second CPs and the various combinations can be screened for their ability to bind the TM.
  • libraries of first CPs and, if desired, also libraries of second CPs in the screening methods provided herein to further increase the complexities of the libraries screened.
  • the libraries screened in these methods is not limited, and the size of the library of first CPs relative to the library of second CPs also is not limited and can be selected as desired by one skilled in the art.
  • the number of members of each of the two or more libraries used can be two or more, but typically can be at least, or at least about 10, 20, 30, 40, 50, 60, 70, 80, 100, 150, 200, 300, 400, 500, 750, 1000, 2000, 5000, 10,000, 50,000, 10 5 , 10 6 , 10 7 , 10 8 , or can fall within a range defined by any two of these values.
  • the two libraries used are a first library of 10 7 Fab light chains and a second library of 10 7 Fab heavy chains, thereby capable in theory of generating and screening a combinatorial library of 10 14 antibodies.
  • the first and second NAM:NABD-CP complexes are contacted with a TM, and at least the first or the second NAM is isolated.
  • the step of contacting the first and second NAM:NABD-CP complexes with the TM is not limited, and can be performed in any sequence as desired by one skilled in the art.
  • a first NAM:NABD-CP complex can be contacted with a TM prior to contacting the first NAM:NABD-CP complex or TM with the second NAM:NABD-CP complex.
  • the first and second NAM:NABD-CP complexes are contacted prior to contacting either complex with the TM.
  • the first and second NAM:NABD-CP complexes and the TM are contacted substantially simultaneously.
  • the order of adding the components of the screen can be selected by one skilled in the art according to the nature of the CPs and TM used, but is typically not limited.
  • the isolation step at least the first or the second NAM that encodes a respective CP that form a (NAM:NABD-CP) 2 :TM complex is isolated from the NAMs that encode CPs that do not form a TM-bound complex.
  • both the first and second NAMs of a TM-bound complex are isolated.
  • the first and second NAMs can be isolated separately or can be isolated together. Separate isolation in the present context does not require separation of each individual NAM from any other NAM, but instead refers to isolation of each NAM (in the same or different samples) without attempting to keep together the first and second NAMs of the same (NAM:NABD-CP) 2 :TM complex.
  • first and second NAMs can be performed by any nucleic acid isolation method and is not particularly limited.
  • the first and second NAMs of each of one or more different (NAM:NABD-CP) 2 :TM complexes are isolated together to the exclusion of other first and second NAMs, thus preserving the coupling of the first and second NAMs that formed each particular (NAM:NABD-CP) 2 :TM complex.
  • Isolation of first and second NAMs from the same complex can be performed by maintaining the complexes intact while isolating the TM-bound complexes from the non-bound complexes.
  • the TM bound complexes are separated from each other by any of a variety of known methods.
  • each TM-bound complex can be transfected into competent cells under conditions that maintain the association of the first and second NAM:NABD-CP complexes in tact under conditions in which substantially only one (NAM:NABD-CP) 2 complex is introduced into any particular cell, which can, but need not include a step of separating the first and second NAM:NABD-CP complexes from the TM.
  • Competent cells can then be selected for the presence of both NAMs and grown as individual colonies, which can be separately treated in subsequent steps such as, validation, further refinement cycles, sequencing, or others as provided elsewhere herein.
  • Such a library can comprise a plurality of nucleic acid molecules containing a target sequence of a NABD and a sequence encoding different fusion proteins that contain a NABD and different CPs, wherein said NABD is capable of binding to the target sequence of the NABD.
  • a library can comprise a plurality of nucleic acid molecules containing a Zn-finger target sequence and a sequence encoding different fusion proteins that contain a Zn-finger binding domain and different candidate polypeptides, wherein said Zn- finger binding domain is capable of binding to the Zn-finger target sequence.
  • Such a library also can comprise a plurality of first nucleic acid molecules, each comprising a first nucleic acid binding domain target sequence and a sequence encoding different first fusion proteins that contains a first nucleic acid binding domain and different first candidate polypeptides, wherein the first nucleic acid binding domain is capable of binding to said first nucleic acid binding domain target sequence; and a second nucleic acid molecule comprising a second nucleic acid binding domain target sequence and a sequence encoding a second fusion protein that contains a second nucleic acid binding domain and a second candidate polypeptide, wherein said second nucleic acid binding domain is capable of binding to the second nucleic acid binding domain target sequence, and wherein the second fusion protein is capable of binding to the first fusion protein.
  • the library further comprises a plurality of different second nucleic acid molecules, each comprising a nucleotide sequence encoding a different second candidate polypeptide, forming a library of second nucleic acid molecules comprising different second candidate polypeptide-encoding sequences.
  • libraries comprising: a plurality nucleic acid molecule-fusion protein complexes comprising: (a) a plurality of nucleic acid molecules containing a target sequence of a nucleic acid binding domain and a sequence encoding different fusion proteins that contain a nucleic acid binding domain and different candidate polypeptides, wherein the nucleic acid binding domain is capable of specifically binding to the target sequence of the nucleic acid binding domain; and (b) the fusion proteins bound to the nucleic acid molecules.
  • libraries include libraries comprising: a plurality nucleic acid molecule-fusion protein complexes comprising: (a) a plurality of nucleic acid molecules containing a Zn-finger target sequence and a sequence encoding different fusion proteins that contain a Zn-finger binding domain and different candidate polypeptides, wherein the Zn-finger binding domain is capable of specifically binding to the Zn-finger target sequence; and (b) the fusion proteins bound to the nucleic acid molecules.
  • the libraries provided herein also can comprise (a) a plurality of first nucleic acid molecule-fusion protein complexes comprising: (i) a plurality of first nucleic acid molecules, each containing a first nucleic acid binding domain target sequence and a sequence encoding different first fusion proteins that contains a first nucleic acid binding domain and different first candidate polypeptides, wherein the first nucleic acid binding domain is capable of specifically binding to said first nucleic acid binding domain target sequence; and (ii) the first fusion proteins bound to the first nucleic acid molecules; and (b) a second nucleic acid molecule-fusion protein complex comprising: (i) a second nucleic acid molecule containing a second nucleic acid binding domain target sequence and a sequence encoding a second fusion protein that contains a second nucleic acid binding domain and a second candidate polypeptide, wherein the second nucleic acid binding domain is capable of specifically binding to the second nucleic acid binding domain target sequence, and wherein said the
  • the second nucleic acid molecule-fusion protein complex further comprises a plurality of different second nucleic acid molecule-fusion protein complexes, each comprising a different second candidate polypeptide, forming a library of second nucleic acid molecule-fusion protein complexes comprising different second candidate polypeptides.
  • kits comprising a nucleic acid molecule comprising a target sequence of a nucleic acid binding domain and a sequence encoding a fusion protein that contains a nucleic acid binding domain and a candidate polypeptide, wherein the nucleic acid binding domain is capable of binding to the target sequence of the nucleic acid binding domain.
  • a kit can comprise a nucleic acid molecule comprising a Zn-finger target sequence and a sequence encoding a fusion protein that contains a Zn-finger binding domain and a candidate polypeptide, wherein the Zn-finger binding domain is capable of binding to the Zn-finger target sequence.
  • kits comprising: (a) a first nucleic acid molecule comprising a first nucleic acid binding domain target sequence and a sequence encoding a first fusion protein that contains a first nucleic acid binding domain and a first candidate polypeptide, wherein the first nucleic acid binding domain is capable of binding to said first nucleic acid binding domain target sequence; and (b) a second nucleic acid molecule comprising a second nucleic acid binding domain target sequence and a sequence encoding a second fusion protein that contains a second nucleic acid binding domain and a second candidate polypeptide, wherein the second nucleic acid binding domain is capable of binding to the second nucleic acid binding domain target sequence, and wherein the second fusion protein is capable of binding to said first fusion protein.
  • Some such kits further comprise a target molecule, which in some embodiments is attached to a detectable moiety or an isolatable moiety.
  • Kits are packaged in combinations that optionally include other reagents or devices for performing screening methods.
  • a kit optionally includes one or more devices for obtaining and manipulating a sample (e.g., competent cells for transfecting CP-encoding NAMs).
  • a kit contains two or more components used in performing a screening method, such as, for example, competent cells, compounds for selecting or inducing transformed cells, reagents for cell lysis, a TM, and reagents or tools for isolating a NAM.
  • the packaging material used in the kit can be one or more physical structures used to house the contents of the kit and can be constructed by well known methods, typically to provide a sterile, contaminant-free environment.
  • the packaging material can have a label that indicates the components of the kit.
  • the packaging material contains instructions indicating how the materials within the kit are employed to perform the screening methods. Instructions typically include a tangible expression describing the reagent concentration or at least one assay method parameter, such as the relative amounts of reagent and sample to be admixed, maintenance time periods for reagent/sample admixtures, temperature, buffer conditions and other parameters.
  • the kit can include one or more containers capable of holding within fixed limits a NAM, NAM:CP complex, a TM, or other reactant or buffer solution used in the screening methods.
  • a kit can include a glass vial used to contain milligram quantities of a NAM, NAM:CP complex, or a TM.
  • a kit also can include substrates, supports or containers for performing the screening methods, including vials, tubes, multi-well plates and/or microarrays.
  • the library plasmid pBAD ZF was constructed in several steps using plasmid pBAD/glll (Invitrogen, Carlsbad, CA) as the starting plasmid.
  • the polylinker region of pBAD/glll was removed by restriction endonuclease cleavage with Ndel and Pmel.
  • a cassette (see Figure 5) containing a polynucleotide encoding the zinc finger protein of SEQ ID NO:2 and additionally encoding a Flag tag (SEQ ID NO: 10) and a His tag (SEQ ID NO: 11), the cassette having (SEQ ID NO:4) was inserted into the Ndel/Pmel site.
  • the plasmid was then transformed into XLl-Blue E. coli cells (Stratagene, San Diego, CA), and the cells were grown and harvested according to the manufacturer's instructions. The plasmid was recovered using the Qiaprep Spin Miniprep Kit (Qiagen, Valencia, CA), and the properly inserted plasmid was isolated using agarose gel electrophoresis. [0105] Next, the zinc finger binding domain sequence (SEQ ID N0:5) was inserted into the plasmid after the terminator by QuikChangeTM site-directed mutagenesis (Stratagene). The final product vector, pBAD ZF (SEQ ID NO: 6), was used for cloning and expression of antibody light chain and heavy chain, as described below. The pBAD ZF was then amplified and isolated using XLl-Blue or DHlOB cells and related methods described above.
  • Antibody light chains and heavy chains were cloned into Ndel and Pad to form pBAD Fab ZF.
  • An exemplary pBAD Fab ZF was formed by digesting pBAD ZF with Ndel and Pad .
  • a cassette containing a nucleotide sequence encoding a light chain antibody domain, a linker, and a heavy chain antibody domain which is inserted so that the heavy chain antibody domain is fused to the N-terminus of the zinc finger protein when translated.
  • the linker serves to link the transcript encoding the light chain and heavy chain/zinc finger proteins, but does not encode a translated polypeptide linker.
  • the light chain and heavy chain/zinc finger proteins are expressed as separate polypeptide chains.
  • the pBAD Fab ZF was then amplified and isolated using XLl-Blue or DHlOB cells and related methods described above.
  • Exemplary plasmids are pBAD_ZF_33389Fab33 (SEQ ID NO:8, Figure 8) and pBAD_ZF_3889Fab35 (SEQ ID NO:9, Figure 9).
  • the library plasmid pBAD33_ZF was constructed in several steps using plasmid pBAD33 (ATCC 87402) as the starting plasmid. First a polylinker, which included RBS and a Ndel site, was introduced. The polylinker region of the plasmid was removed by restriction endonuclease cleavage using Ndel and Hindi ⁇ A cassette (purified from pBAD ZF) containing a polynucleotide encoding the zinc finger protein of SEQ ID NO:2 and additionally encoding a Flag tag and a His tag (the cassette having SEQ ID NO:4) was inserted into the Ndel/Hindl site. The properly inserted plasmid was then amplified and isolated using XLl-Blue cells and related methods described above.
  • the zinc finger binding domain sequence (SEQ ID NO: 3) was inserted into the vector after the terminator by QuikChangeTM site-directed mutagenesis.
  • the final product vector, pBAD33_ZF (SEQ ID NO: 7), was used for cloning and expression of antibody light chain or heavy chain, as described below.
  • the pBAD33_ZF was then amplified and isolated using XLl-Blue or DHlOB cells and related methods described above.
  • Antibody light chains or heavy chains were cloned into Ndel and Pad to form pBAD33_LC_ZF or pBAD33_HC_ZF, respectively.
  • An exemplary pBAD33_LC_ZF was formed by digesting pBAD33_ZF with Ndel and Pad. Into the Ndel/Pacl site was inserted a cassette containing a nucleotide sequence encoding a light chain antibody domain, which is inserted so that the light chain antibody domain is fused to the N-terminus of the zinc finger protein when translated.
  • An exemplary pBAD33_HC_ZF was formed by digesting pBAD33_ZF with Ndel and Pad Into the Ndel/Pacl site was inserted a cassette containing a nucleotide sequence encoding a heavy chain antibody domain, which is inserted so that the heavy chain antibody domain is fused to the N-terminus of the zinc finger protein when translated.
  • the pBAD33_LC_ZF and pBAD33_HC_ZF were then amplified and isolated using XLl-Blue or DHlOB cells and related methods described above.
  • cDNA from immunized mice was obtained commercially and libraries were constructed using the BD SMARTTM kit according to the manufacturer's instructions (BD Biosciences, San Jose, CA).
  • cDNA encoding light chain antibody domains was amplified using 11 V L forward primers and a kappa reverse primer.
  • cDNA encoding heavy chain antibody domains was amplified using 11 V H forward primers and 6 reverse primers to complement IgGl-I, IgGl -2, IgG2a, IgG2c, IgG2c-2 and IgG3..
  • Heavy chain PCR products were cloned into expression vector pBAD33_ZF according to the procedure described above, to yield a library of plasmids pBAD33_HC_ZF.
  • the library of plasmids was transformed into expression host Rosetta- gamiTM(DE3) Competent Cells (Novagen, Madison, WI) cells according to the manufacturer's instructions.
  • a stock of transformed expression host cells was prepared and maintained as described for the DNA Display libraries.
  • Magnetic Dyna beads (M270 Epoxy 142.01) (30 mg) were washed in ImI of 0.1M Phosphate buffer (pH 8.0). The beads were placed on a magnetic stand and the supernatant was removed. Wash was repeated 2-3 more times and the beads were resuspended in a final volume of 500 ⁇ l of wash buffer. The beads were then mixed overnight with 500 ⁇ l of approximately 1 mg/ml antigen. For example, using SARS spike protein as an exemplary antigen, the beads were then mixed overnight with 500 ⁇ l of approximately 1 mg/ml of SARS spike protein. The beads were then washed twice with wash buffer and then resuspended in 1 ml of wash buffer.
  • the cells were grown at 37 0 C for 2hrs (until middle log phrase), and then induced for 3-12 hrs at 2O 0 C by adding arabinose (0.2% final concentration) and ZnCl 2 (10OmM final concentration). The induced cells were then centrifuged for 15 min at 5000 rpm in a Beckmann JA- 12 conical rotor, and the supernatant was removed.
  • the cell pellet was resuspended in sonication buffer (0.5 mM DTT, 0.05% Tween 20, 0.1 mg/ml ssDNA (Salmon sperm DNA), 10 mg/ml BSA (or 5% milk), 50 mM Na-Glutamate, 100 ⁇ M ZnC12, 10 mM HEPES, pH 7.4), centrifuged, and again resuspended in 250 - 500 ⁇ l of sonication buffer. On ice, the cells were sonicated 6 sec at setting 3 in a Sonicator. Samples were then centrifuged at 4 0 C.
  • sonication buffer 0.5 mM DTT, 0.05% Tween 20, 0.1 mg/ml ssDNA (Salmon sperm DNA), 10 mg/ml BSA (or 5% milk), 50 mM Na-Glutamate, 100 ⁇ M ZnC12, 10 mM HEPES, pH 7.4
  • Antigen-labeled magnetic beads (100 ⁇ l) were washed with 2 x fresh sonication buffer, added to the cell lysate described above, and mixed for at least 45 min at room temperature. On a magnetic stand, supernatant was removed and the beads were washed with 1 ml of sonication buffer, then washed with 1 ml elution buffer (0.5 mM DTT, 0.05% Tween 20, 10 mg/ml BSA, 50 mM Na-Glutamate, 100 ⁇ M ZnC12, 10 mM HEPES, pH 7.4). The beads were then centrifuged to remove remaining buffer.
  • the beads were mixed for 5 minutes with 50 ⁇ l of elution buffer + 0.5M NaCl (final concentration), then supernatant was removed, which eluted the bound plasmids.
  • the eluted plasmid DNA was cleaned using Roche High Pure PCR Product Purification Kit (Roche Diagnostics, Manheim, Germany). The clean DNA was then transformed into high transformation efficiency DHlOB or XIl -blue E. coli cells by electroporation according to manufacturer's instructions. The transformed cells were then grown on LB plates with antibiotics at 30 0 C and harvested. The plasmid DNA was then recovered using a Qiaprep Spin Miniprep Kit (Qiagen, Valencia, CA). Secondary screen
  • Recovered plasmids were retransformed into the same expression strain as described above. Approximately 1000-10,000 single colonies were selected and pooled into 5 ml LB containing carbenicillin (100 ⁇ g/ml final concentration). The cells were grown and induced as described above. The secondary screen was done using the method provided above.
  • Each putative hit was purified, expressed and re-assayed using the methods provided below. It can be done after primary screen or after secondary screen. Recovered plasmids were retransformed into the same expression strain as described above. Approximately 100-500 single colonies were selected and placed into duplicate 96-well plates containing 0.2 ml LB containing carbenicillin (100 ⁇ g/ml final concentration). The cells were grown for 16 hrs, and then diluted 50-100 fold into ImI LB containing carbenicillin. The diluted cells were grown and induced at middle log phrase with arabinose (0.2% final concentration) and ZnCl 2 (10OmM final concentration) for 3-12hrs at 2O 0 C.
  • the induced cells in 96-well plates were then centrifuged for 20 min at 4000 rpm in a centrifuge.
  • the pellets were resuspended in 125 ⁇ l of sonication buffer, and transferred to a Fisher skirted 96-well PCR plate (Fisher Scientific, Pittsburgh, PA), and the plate was sealed with an aluminum seal.
  • the cells were then sonicated 6x for 1 min at an output setting of 5.0 in a ice-bath chilled Misonix Sonicator Microplate horn (Misonix, Farmingdale, NY).
  • the plates were then centrifuged at 1000 rpm for 25 min and the lysate was added directly onto an antigen coated 96-well ELISA plate (see below).
  • the antigen coated 96-well ELISA plate was prepared by incubating 100 ⁇ l of antigen (1 ⁇ g/ml) to each well at 4 0 C for overnight, and then washing the plates with 200 ⁇ l/well of PBS plus 0.1% Tween 20 (PBS-T).
  • An exemplary antigen coated 96-well ELISA plate was prepared by incubating 100 ⁇ l of SARS spike protein (1 ⁇ g/ml) to each well at 4 0 C for overnight, and then washing the plates with 200 ⁇ l/well of PBS plus 0.1% Tween 20 (PBS-T).
  • Magnetic Dyna beads were prepared as described in Example 3, where the beads were mixed overnight with 500 ⁇ l of approximately 1 mg/ml of the exemplary antigen lysozyme.

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Abstract

L'invention concerne des molécules d'acide nucléique ayant des séquences cibles qui peuvent être liées par des protéines de fusion, qui sont codées par d'autres séquences dans les mêmes molécules d'acide nucléique. L'invention concerne en outre des procédés d'isolement de séquences d'acide nucléique codant pour des polypeptides qui se lient à une molécule cible.
PCT/US2007/080350 2006-10-04 2007-10-03 Ecran d'affichage d'adn pour un produit d'expression avec des propriétés de liaison désirées WO2008140538A1 (fr)

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Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2013053719A3 (fr) * 2011-10-11 2013-06-27 Aliophtha Ag Régulation de l'expression d'un récepteur par l'intermédiaire de l'administration de facteurs de transcription artificiels
CN104220458A (zh) * 2012-02-24 2014-12-17 阿特根公司 结合有含半胱氨酸残基的基序的修饰抗体，含该修饰抗体的修饰抗体-药物缀合物以及其制造方法
CN112135906A (zh) * 2018-04-23 2020-12-25 格扎Ad有限公司 被衣壳单元包封的核酸折纸结构

Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20040197892A1 (en) * 2001-04-04 2004-10-07 Michael Moore Composition binding polypeptides
US20050191662A1 (en) * 2003-12-22 2005-09-01 Syngenta Participations Ag In vitro screening and evolution of proteins
US20060029947A1 (en) * 2004-03-18 2006-02-09 Board Of Regents, The University Of Texas System Combinatorial protein library screening by periplasmic expression

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20040197892A1 (en) * 2001-04-04 2004-10-07 Michael Moore Composition binding polypeptides
US20050191662A1 (en) * 2003-12-22 2005-09-01 Syngenta Participations Ag In vitro screening and evolution of proteins
US20060029947A1 (en) * 2004-03-18 2006-02-09 Board Of Regents, The University Of Texas System Combinatorial protein library screening by periplasmic expression

Cited By (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2013053719A3 (fr) * 2011-10-11 2013-06-27 Aliophtha Ag Régulation de l'expression d'un récepteur par l'intermédiaire de l'administration de facteurs de transcription artificiels
CN104220458A (zh) * 2012-02-24 2014-12-17 阿特根公司 结合有含半胱氨酸残基的基序的修饰抗体，含该修饰抗体的修饰抗体-药物缀合物以及其制造方法
JP2015513541A (ja) * 2012-02-24 2015-05-14 アルテオジェン インコーポレイテッドＡｌｔｅｏｇｅｎ Ｉｎｃ． システイン残基を含むモチーフが結合した修飾抗体、前記修飾抗体を含む修飾抗体−薬物複合体及びその製造方法
EP2818480A4 (fr) * 2012-02-24 2015-10-07 Alteogen Inc Anticorps modifié dans lequel un motif comportant un résidu cystéine est lié, conjugué anticorps modifié-médicament comportant l'anticorps modifié, et son procédé de fabrication
US9814782B2 (en) 2012-02-24 2017-11-14 Alteogen Inc. Modified antibody in which motif comprising cysteine residue is bound, modified antibody-drug conjugate comprising the modified antibody, and production method for same
CN104220458B (zh) * 2012-02-24 2018-08-14 阿特根公司 结合有含半胱氨酸残基的基序的修饰抗体，含该修饰抗体的修饰抗体-药物缀合物以及其制造方法
CN112135906A (zh) * 2018-04-23 2020-12-25 格扎Ad有限公司 被衣壳单元包封的核酸折纸结构

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