METHODS AND COMPOSITIONS FOR PRODUCING A CHIMERIC POLYPEPTIDE

The present invention provides methods and compositions for converting a first polypeptide into a chimeric polypeptide. The invention includes two vectors: a first vector including the sequence of the first polypeptide and a second vector including a second polypeptide. The vectors include complementary site-specific recombination motifs such that site-specific recombination between the two vectors results in the generation of a chimeric polypeptide including at least a portion of the first polypeptide and at least a portion of the second polypeptide. A site-specific recombination motif may be positioned within an intron or within a coding sequence on the first or second vector.

METHODS FOR THE PRODUCTION OF LIBRARIES FOR DIRECTED EVOLUTION

Disclosed herein is an efficient method of generating a library of variants of a sequence of interest, such as may be used in directed evolution, in one embodiment, the method includes an amplification reaction, e.g. error-prone PCR, to generate double-stranded DNA (dsDNA) variants of a sequence of interest, after which one strand of the dsDNA variants may be selectively degraded to produce single-stranded DNA (ssDNA) variants. The ssDNA variants may be hybridized to ssDNA intermediaries, e.g., uracilated circular ssDNA intermediaries, to form heteroduplex DNA, which may be transformed into cells, such as cells, yielding a library of variants. This method eliminates the inefficient sub-cloning steps and the need for costly primer sets required by many prior methods. 1. A method of generating a library of variants of a sequence of interest , said method comprising the steps of:(a) providing a template DNA molecule comprising said sequence of interest;(b) providing a pair of oligonucleotides wherein said oligonucleotides hybridize to opposite strands of said sequence of interest, wherein one of said oligonucleotides is protected, the other oligonucleotide is non-protected, and said oligonucleotides flank said sequence of interest;(c) performing an amplification reaction on said template DNA molecule using said oligonucleotides, thereby generating a population of dsDNA variants;(d) incubating said population of dsDNA variants with an enzyme capable of selectively degrading the non-protected strand over the protected strand of said dsDNA variants, thereby producing a population of ssDNA variants;(e) hybridizing said population of ssDNA variants to ssDNA intermediaries, wherein said ssDNA intermediaries comprise a sequence substantially identical to said sequence of interest or a fragment thereof, generating heteroduplex DNA; and(f) transforming said heteroduplex DNA into cells, thereby generating a library of variants of said sequence of interest.2. The method of claim ...

METHODS OF UTILIZING RECOMBINATION FOR THE IDENTIFICATION OF BINDING MOIETIES

The identification of binding moieties capable of selectively interacting with one or more target antigens is of scientific, medical, and commercial value. Disclosed herein are methods and compositions for the identification, labeling, and/or retrieval of cognate binding moieties. 1. A method of identifying a cognate binding moiety of one or more antigens , said method comprising the steps of: [ i) a cell surface antigen and', 'ii) a nucleic acid comprising a first recombination motif, and, '1) a population of cells, each of said cells comprising, i) one of a plurality of transgenic viral surface binding moieties and', 'ii) a nucleic acid comprising a second recombination motif capable of integrating with said first motif;, '2) a plurality of attachment-defective virions, each of said virions comprising], 'a) providing'}b) contacting said population of cells with said virions, wherein, if one or more of said virions comprise a cognate binding moiety of one or more of said cell surface antigens, binding of said cell surface antigen and said cognate binding moiety results in selective infection by the bound virion of the cell comprising said cell surface antigen of said cognate binding moiety;c) incubating said population of cells under conditions sufficient to allow recombination between said first and second recombination motifs in infected cells, thereby generating a recombinant product; andd) identifying a cognate binding moiety of one or more of said antigens by identifying the nucleic acid sequence of said recombinant product or an identifying fragment thereof, in a cell, and identifying from said nucleic acid sequence an antigen and a cognate binding moiety.2. The method of claim 1 , wherein said cell comprises a recombination enzyme.3. The method of or claim 1 , wherein the nucleic acid comprised by one or more of said virions encodes a recombination enzyme and said incubation is sufficient to allow expression of said encoded recombination enzyme.4. The method ...

COMPOSITIONS AND METHODS FOR THE IDENTIFICATION AND ISOLATION OF CELL-MEMBRANE PROTEIN SPECIFIC BINDING MOIETIES

Disclosed are methods of identifying binding moieties that recognize antigens displayed on cells, such as membrane proteins or recombinant proteins that display eptiopes on the surface of cells. Binding moieties capable of binding membrane proteins can be difficult to obtain because these proteins can depend on their native environments for structural integrity. In some methods scFv phage display libraries are panned against whole cells expressing a membrane protein in an emulsion. Certain methods further permit discrimination of binding moieties according to their affinity or avidity for a target. This approach allows rapid identification of cell surface epitope specific antibodies for research, diagnostics, and immunotherapeutics. 114-. (canceled)15. An emulsion comprising a plurality of droplets , wherein said droplets comprise:(i) a plurality of antigen carriers displaying a cell-membrane bound epitope, wherein said antigen carriers comprise animal cells; and(ii) a plurality of binding moiety carriers, wherein the plurality of said binding moiety carriers comprise viruses encoding and displaying the binding moieties, the viruses being released into said droplets by virus-transduced bacteria.16. The emulsion of claim 15 , wherein claim 15 , within said droplets claim 15 , one or more of said antigen carriers displaying said cell-membrane bound epitope contacts a plurality of said binding moiety carriers; andwherein, if an individual binding moiety is specific to said antigen carrier present in the same droplet, said contacting results in a complex comprising at least one binding moiety bound to a membrane of an intact binding moiety carrier and at least one antigen bound to a membrane of an intact antigen carrier.17. The emulsion of claim 15 , wherein said binding moiety carrier is an M13 bacteriophage claim 15 , a lambda phage claim 15 , a T4 phage claim 15 , or a T7 phage.18. The emulsion of claim 15 , wherein said binding moiety comprises a DARPin claim 15 , ...

METHODS AND COMPOSITIONS FOR PRODUCING A CHIMERIC POLYPEPTIDE

The present invention provides methods and compositions for converting a first polypeptide into a chimeric polypeptide. The invention includes two vectors: a first vector including the sequence of the first polypeptide and a second vector including a second polypeptide. The vectors include complementary site-specific recombination motifs such that site-specific recombination between the two vectors results in the generation of a chimeric polypeptide including at least a portion of the first polypeptide and at least a portion of the second polypeptide. A site-specific recombination motif may be positioned within an intron or within a coding sequence on the first or second vector. 1. A method of converting a single-chain variable fragment (scFv) into a chimeric polypeptide , comprising: a first mammalian expression control motif,', {'i': 'E. coli', 'a first expression control motif,'}, 'a sequence encoding a heavy chain variable region (VH) of the scFv,', 'a first site-specific recombination motif,', 'a sequence encoding a light chain variable region (VL) of the scFv,', {'sub': '5′SS', 'a 5′ mammalian splice site (Mam),'}, 'a fusion display protein sequence,', {'sub': '3′SS', 'a 3′ mammalian splice site (Mam), and'}, 'a sequence encoding a light chain constant region (CL), and, '(a) providing a first vector comprising, in order from 5′ to 3′,'} a second mammalian expression control motif,', 'a second site-specific recombination motif, and', 'a sequence encoding a polypeptide; and, '(b) providing a second vector comprising, in order from 5′ to 3′,'}(c) contacting the first vector and the second vector in the presence of a recombinase enzyme,wherein the recombinase enzyme combines the first vector and the second vector in a site-specific manner to form an integrant vector, andwherein the integrant vector expresses the VL fused to the CL and a separate VH fused to said polypeptide,thereby upon expression converting an scFv into a chimeric polypeptide.2. The method of ...

METHODS OF UTILIZING RECOMBINATION FOR THE IDENTIFICATION OF BINDING MOIETIES

The identification of binding moieties capable of selectively interacting with one or more target antigens is of scientific, medical, and commercial value. Disclosed herein are methods and compositions for the identification, labeling and/or retrieval of cognate binding moieties. 145-. (canceled)46. A method of identifying a cognate binding moiety of one or more antigens , said method comprising the steps of: [ i) a cell surface antigen,', 'ii) a nucleic acid comprising a first recombination motif, and', 'iii) a conditionally expressed infection apparatus; and, '1) a population of cells, each of said cells comprising, i) one of a plurality of transgenic viral surface binding moieties and', 'ii) a nucleic acid comprising a second recombination motif capable of integrating with said first motif;, '2) a plurality of virions, each of said virions comprising], 'a) providing'}b) contacting said population of cells with said virions;c) incubating said cells with said virions under conditions that are not permissive for infection but are sufficient to allow binding of one or more of said cell surface antigens by one or more of said cognate binding moieties, whereby one or more of said cells are bound by one or more of said virions;d) emulsifying said bound cells, thereby encapsulating one or more of said cells in one or more emulsion droplets;e) incubating said emulsified cells under conditions permissive to expression of said conditionally expressed infection apparatus, thereby allowing infection of one or more of said emulsified cells by one or more virions present in the same emulsion droplet;f) incubating said cells in a manner sufficient to allow recombination between said first and second site-specific recombination motifs in infected cells, thereby generating a recombinant product; andg) identifying a cognate binding moiety of one or more of said antigens by identifying the nucleic acid sequence of said recombinant product or an identifying fragment thereof, in a ...

METHODS FOR THE PRODUCTION OF LIBRARIES FOR DIRECTED EVOLUTION

Disclosed herein is an efficient method of generating a library of variants of a sequence of interest, such as may be used in directed evolution, in one embodiment, the method includes an amplification reaction, e.g. error-prone PCR, to generate double-stranded DNA (dsDNA) variants of a sequence of interest, after which one strand of the dsDNA variants may be selectively degraded to produce single-stranded DNA (ssDNA) variants. The ssDNA variants may be hybridized to ssDNA intermediaries, e.g., uracilated circular ssDNA intermediaries, to form heteroduplex DNA, which may be transformed into cells, such as cells, yielding a library of variants. This method eliminates the inefficient sub-cloning steps and the need for costly primer sets required by many prior methods. 1. A method of generating a library of variants of a sequence of interest , said method comprising the steps of:(a) providing a template DNA molecule comprising said sequence of interest;(b) providing a pair of oligonucleotides wherein said oligonucleotides hybridize to opposite strands of said sequence of interest, wherein one of said oligonucleotides is protected, the other oligonucleotide is non-protected, and said oligonucleotides flank said sequence of interest;(c) performing an amplification reaction on said template DNA molecule using said oligonucleotides, thereby generating a population of dsDNA variants;(d) incubating said population of dsDNA variants with an enzyme capable of selectively degrading the non-protected strand over the protected strand of said dsDNA variants, thereby producing a population of ssDNA variants;(e) hybridizing said population of ssDNA variants to ssDNA intermediaries, wherein said ssDNA intermediaries comprise a sequence substantially identical to said sequence of interest or a fragment thereof, generating heteroduplex DNA; and(f) transforming said heteroduplex DNA into cells, thereby generating a library of variants of said sequence of interest.2. The method of claim ...

METHODS FOR THE PRODUCTION OF LIBRARIES FOR DIRECTED EVOLUTION

Disclosed herein is an efficient method of generating a library of variants of a sequence of interest, such as may be used in directed evolution, in one embodiment, the method includes an amplification reaction, e.g. error-prone PCR, to generate double-stranded DNA (dsDNA) variants of a sequence of interest, after which one strand of the dsDNA variants may be selectively degraded to produce single-stranded DNA (ssDNA) variants. The ssDNA variants may be hybridized to ssDNA intermediaries, e.g., uracilated circular ssDNA intermediaries, to form heteroduplex DNA, which may be transformed into cells, such as cells, yielding a library of variants. This method eliminates the inefficient sub-cloning steps and the need for costly primer sets required by many prior methods. 1. A method of generating a library of variants of a sequence of interest , said method comprising the steps of:(a) providing a template DNA molecule comprising said sequence of interest;(b) providing a pair of oligonucleotides wherein said oligonucleotides hybridize to opposite strands of said sequence of interest, wherein one of said oligonucleotides is protected, the other oligonucleotide is non-protected, and said oligonucleotides flank said sequence of interest;(c) performing an amplification reaction on said template DNA molecule using said oligonucleotides, thereby generating a population of dsDNA variants;(d) incubating said population of dsDNA variants with an enzyme capable of selectively degrading the non-protected strand over the protected strand of said dsDNA variants, thereby producing a population of ssDNA variants;(e) hybridizing said population of ssDNA variants to ssDNA intermediaries, wherein said ssDNA intermediaries comprise a sequence substantially identical to said sequence of interest or a fragment thereof, generating heteroduplex DNA; and(f) transforming said heteroduplex DNA into cells, thereby generating a library of variants of said sequence of interest.2. The method of claim ...

METHODS AND COMPOSITIONS FOR THE DEVELOPMENT OF ANTIBODIES SPECIFIC TO EPITOPE POST-TRANSLATIONAL MODIFICATION STATUS

The present disclosure provides, among other things, a method of generating antibodies that recognize a protein of interest. In some aspects, the protein of interest contains a post translational modification (PTM) site. Provided in some aspects is a method of generating non-PTM-binding antibodies that specifically bind a site without post translational modification. Provided in some aspects is a pan-PTM-binding antibody library comprising a plurality of antibodies derived from a pre-existing antibody that specifically recognizes a PTM on a peptide or protein of interest. Provided in further aspects is a non-PTM-binding antibody library comprising a plurality of antibodies derived from a pre-existing antibody that specifically recognizes a PTM on a peptide or protein of interest. 122-. (canceled)23. A method of generating a pair of modification-specific antibody clonotypes comprising:(a) providing an antibody that specifically recognizes a PTM on a peptide or protein of interest;identifying a PTM binding pocket of the antibody;(b) introducing an amino acid that repels the PTM at one or more sites in the PTM binding pocket of the antibody that are determined to interact with the PTM;(c) generating a library comprising candidate non-PTM binding antibodies by randomizing one or more regions outside the PTM binding pocket that bind to a context sequence adjacent to the PTM site;(d) screening the library against the peptide or protein of interest without PTM, and(e) selecting a non-PTM binding antibody to provide a pair of PTM-status-specific antibody clonotypes.24. The method of claim 23 , wherein the PTM site is a naturally occurring PTM site.2530-. (canceled)31. The method of claim 23 , wherein the one or more sites within the PTM binding pocket are structurally-predicted.32. The method of claim 23 , wherein the one or more sites within the PTM binding pocket are experimentally-determined.33. (canceled)34. The method of claim 23 , wherein the PTM is phosphorylation ...

Methods and compositions for ligand directed antibody design

The present disclosure provides, among other things, methods for generating antibodies against a target protein. In some embodiments, a library is provided comprising a plurality of tether antibodies comprising an antigen binding region and a ligand that binds to a target protein. In some embodiments, a library is provided comprising a plurality of candidate antibodies for binding to a target protein.

METHODS FOR THE PRODUCTION OF LIBRARIES FOR DIRECTED EVOLUTION

Disclosed herein is an efficient method of generating a library of variants of a sequence of interest, such as may be used in directed evolution. In one embodiment, the method includes an amplification reaction, e.g., error-prone PCR, to generate double-stranded DNA (dsDNA) variants of a sequence of interest, after which one strand of the dsDNA variants may be selectively degraded to produce single-stranded DNA (ssDNA) variants. The ssDNA variants may be hybridized to ssDNA intermediaries, e.g., uracilated circular ssDNA intermediaries, to form heteroduplex DNA, which may be transformed into cells, such as cells, yielding a library of variants. This method eliminates the inefficient sub-cloning steps and the need for costly primer sets required by many prior methods. 170-. (canceled)71. A method of selectively degrading a non-recombinant nucleic acid , the method comprising:(a) providing a first nucleic acid comprising at least one segment having a restriction site, wherein said at least one segment comprises a sequence encoding a vector;(b) providing at least one second nucleic acid comprising at least one segment that does not comprise said restriction site, wherein said at least one segment comprises a sequence encoding at least one of a scFv light chain variable region, a scFv heavy chain variable region, a scFv linker region, an IgG heavy chain constant region, an IgG light chain constant region, a gpIII protein, and any combination thereof;(c) subjecting said first nucleic acid and said at least one second nucleic acid to a recombination reaction such that said segment of said first nucleic acid is replaced with said segment of said second nucleic acid, thereby generating a recombinant product that does not include said restriction site and a recombinant product that includes said restriction site;(d) transforming the recombination products of said reaction into a cell expressing a restriction enzyme capable of cleaving said restriction site; and(e) incubating ...

COMPOSITIONS AND METHODS FOR THE IDENTIFICATION AND ISOLATION OF CELL-MEMBRANE PROTEIN SPECIFIC BINDING MOIETIES

Disclosed are methods of identifying binding moieties that recognize antigens displayed on cells, such as membrane proteins or recombinant proteins that display eptiopes on the surface of cells. Binding moieties capable of binding membrane proteins can be difficult to obtain because these proteins can depend on their native environments for structural integrity. In some methods scFv phage display libraries are panned against whole cells expressing a membrane protein in an emulsion. Certain methods further permit discrimination of binding moieties according to their affinity or avidity for a target. This approach allows rapid identification of cell surface epitope specific antibodies for research, diagnostics, and immunotherapeutics. 2. The method of claim 1 , wherein said binding moiety carrier is a virus or cell.3. The method of claim 2 , wherein said binding moiety carrier is a lambda phage claim 2 , T4 phage claim 2 , T7 phage claim 2 , yeast cell claim 2 , or bacterial cell.4. The method of claim 2 , wherein said virus or cell comprises a DARPin claim 2 , FN3 (fibronectin repeats) claim 2 , Fork-head associate protein claim 2 , scFv claim 2 , Fab claim 2 , or other antibody-derived protein.5. The method of claim 1 , wherein said binding moiety carrier is released into said individual droplet by virus-transduced bacteria.6. The method of claim 5 , wherein said virus-transduced bacteria are phage-transduced bacteria.7. The method of claim 1 , further comprising repeating steps a-f with a plurality of binding moieties isolated in step f.8. The method of claim 1 , wherein said labeled binding agent comprises a fluorescent label.9. The method of claim 8 , wherein said isolation of step f occurs by fluorescent activated cell sorting (FACS).10. The method of claim 1 , wherein said cell-membrane bound epitope is recombinantly expressed by said cells.11. The method of claim 1 , wherein said cell-membrane bound epitope is natively expressed by said cells.12. The method of ...

Methods of utilizing recombination for the identification of binding moieties

The identification of binding moieties capable of selectively interacting with one or more target antigens is of scientific, medical, and commercial value. Disclosed herein are methods and compositions for the identification, labeling, and/or retrieval of cognate binding moieties.

Methods of utilizing recombination for the identification of binding moieties

The identification of binding moieties capable of selectively interacting with one or more target antigens is of scientific, medical, and commercial value. Disclosed herein are methods and compositions for the identification, labeling and/or retrieval of cognate binding moieties.

Methods and compositions for the development of antibodies specific to epitope post-translational modification status

The present disclosure provides, among other things, a method of generating antibodies that recognize a protein of interest. In some aspects, the protein of interest contains a post translational modification (PTM) site. Provided in some aspects is a method of generating non-PTM-binding antibodies that specifically bind a site without post translational modification. Provided in some aspects is a pan-PTM-binding antibody library comprising a plurality of antibodies derived from a pre-existing antibody that specifically recognizes a PTM on a peptide or protein of interest. Provided in further aspects is a non-PTM-binding antibody library comprising a plurality of antibodies derived from a pre-existing antibody that specifically recognizes a PTM on a peptide or protein of interest.

Methods for the production of libraries for directed evolution

Disclosed herein is an efficient method of generating a library of variants of a sequence of interest, such as may be used in directed evolution, in one embodiment, the method includes an amplification reaction, e.g. error-prone PCR, to generate double-stranded DNA (dsDNA) variants of a sequence of interest, after which one strand of the dsDNA variants may be selectively degraded to produce single-stranded DNA (ssDNA) variants. The ssDNA variants may be hybridized to ssDNA intermediaries, e.g., uracilated circular ssDNA intermediaries, to form heteroduplex DNA, which may be transformed into cells, such as E. coli cells, yielding a library of variants. This method eliminates the inefficient sub-cloning steps and the need for costly primer sets required by many prior methods.

Methods for the production of libraries for directed evolution

Disclosed herein is an efficient method of generating a library of variants of a sequence of interest, such as may be used in directed evolution, in one embodiment, the method includes an amplification reaction, e.g. error-prone PCR, to generate double-stranded DNA (dsDNA) variants of a sequence of interest, after which one strand of the dsDNA variants may be selectively degraded to produce single-stranded DNA (ssDNA) variants. The ssDNA variants may be hybridized to ssDNA intermediaries, e.g., uracilated circular ssDNA intermediaries, to form heteroduplex DNA, which may be transformed into cells, such as

Methods and compositions for the development of antibodies specific to epitope post-translational modification status

The present disclosure provides, among other things, a method of generating antibodies that recognize a protein of interest. In some aspects, the protein of interest contains a post translational modification (PTM) site. Provided in some aspects is a method of generating non-PTM-binding antibodies that specifically bind a site without post translational modification. Provided in some aspects is a pan-PTM-binding antibody library comprising a plurality of antibodies derived from a pre-existing antibody that specifically recognizes a PTM on a peptide or protein of interest. Provided in further aspects is a non-PTM-binding antibody library comprising a plurality of antibodies derived from a pre-existing antibody that specifically recognizes a PTM on a peptide or protein of interest.

Compositions and methods for the identification and isolation of cell-membrane protein specific binding moieties

Compositions and methods for the identification and isolation of cell-membrane protein specific binding moieties

Disclosed are methods of identifying binding moieties that recognize antigens displayed on cells, such as membrane proteins or recombinant proteins that display eptiopes on the surface of cells. Binding moieties capable of binding membrane proteins can be difficult to obtain because these proteins can depend on their native environments for structural integrity. In some methods scFv phage display libraries are panned against whole cells expressing a membrane protein in an emulsion. Certain methods further permit discrimination of binding moieties according to their affinity or avidity for a target. This approach allows rapid identification of cell surface epitope specific antibodies for research, diagnostics, and immunotherapeutics.

Compositions and methods for the identification and isolation of cell-membrane protein specific binding moieties

Disclosed are methods of identifying binding moieties that recognize antigens displayed on cells, such as membrane proteins or recombinant proteins that display eptiopes on the surface of cells. Binding moieties capable of binding membrane proteins can be difficult to obtain because these proteins can depend on their native environments for structural integrity. In some methods scFv phage display libraries are panned against whole cells expressing a membrane protein in an emulsion. Certain methods further permit discrimination of binding moieties according to their affinity or avidity for a target. This approach allows rapid identification of cell surface epitope specific antibodies for research, diagnostics, and immunotherapeutics.

Methods for the production of libraries for directed evolution

Disclosed herein is an efficient method of generating a library of variants of a sequence of interest, such as may be used in directed evolution, in one embodiment, the method includes an amplification reaction, e.g. error-prone PCR, to generate double-stranded DNA (dsDNA) variants of a sequence of interest, after which one strand of the dsDNA variants may be selectively degraded to produce single-stranded DNA (ssDNA) variants. The ssDNA variants may be hybridized to ssDNA intermediaries, e.g., uracilated circular ssDNA intermediaries, to form heteroduplex DNA, which may be transformed into cells, such as E. coli cells, yielding a library of variants. This method eliminates the inefficient sub-cloning steps and the need for costly primer sets required by many prior methods.

Methods and compositions for ligand directed antibody design

The present disclosure provides, among other things, methods for generating antibodies against a target protein. In some embodiments, a library is provided comprising a plurality of tether antibodies comprising an antigen binding region and a ligand that binds to a target protein. In some embodiments, a library is provided comprising a plurality of candidate antibodies for binding to a target protein.

Methods and compositions for ligand directed antibody design

The present disclosure provides, among other things, methods for generating antibodies against a target protein. In some embodiments, a library is provided comprising a plurality of tether antibodies comprising an antigen binding region and a ligand that binds to a target protein. In some embodiments, a library is provided comprising a plurality of candidate antibodies for binding to a target protein.

Methods and compositions for producing a chimeric polypeptide

The present invention provides methods and compositions for converting a first polypeptide into a chimeric polypeptide. The invention includes two vectors: a first vector including the sequence of the first polypeptide and a second vector including a second polypeptide. The vectors include complementary site-specific recombination motifs such that site-specific recombination between the two vectors results in the generation of a chimeric polypeptide including at least a portion of the first polypeptide and at least a portion of the second polypeptide. A site-specific recombination motif may be positioned within an intron or within a coding sequence on the first or second vector.

Methods and compositions for producing a chimeric polypeptide

The present invention provides methods and compositions for converting a first polypeptide into a chimeric polypeptide. The invention includes two vectors: a first vector including the sequence of the first polypeptide and a second vector including a second polypeptide. The vectors include complementary site-specific recombination motifs such that site-specific recombination between the two vectors results in the generation of a chimeric polypeptide including at least a portion of the first polypeptide and at least a portion of the second polypeptide. A site-specific recombination motif may be positioned within an intron or within a coding sequence on the first or second vector.

Methods and compositions for producing a chimeric polypeptide

The present invention provides methods and compositions for converting a first polypeptide into a chimeric polypeptide. The invention includes two vectors: a first vector including the sequence of the first polypeptide and a second vector including a second polypeptide. The vectors include complementary site-specific recombination motifs such that site-specific recombination between the two vectors results in the generation of a chimeric polypeptide including at least a portion of the first polypeptide and at least a portion of the second polypeptide. A site-specific recombination motif may be positioned within an intron or within a coding sequence on the first or second vector.

Methods for the production of libraries for directed evolution

Disclosed herein is an efficient method of generating a library of variants of a sequence of interest, such as may be used in directed evolution. In one embodiment, the method includes an amplification reaction, e.g., error-prone PCR, to generate double-stranded DNA (dsDNA) variants of a sequence of interest, after which one strand of the dsDNA variants may be selectively degraded to produce single-stranded DNA (ssDNA) variants. The ssDNA variants may be hybridized to ssDNA intermediaries, e.g., uracilated circular ssDNA intermediaries, to form heteroduplex DNA, which may be transformed into cells, such as E. coli cells, yielding a library of variants. This method eliminates the inefficient sub-cloning steps and the need for costly primer sets required by many prior methods.

Methods for the production of libraries for directed evolution

Disclosed herein is an efficient method of generating a library of variants of a sequence of interest, such as may be used in directed evolution, in one embodiment, the method includes an amplification reaction, e.g. error-prone PCR, to generate double-stranded DNA (dsDNA) variants of a sequence of interest, after which one strand of the dsDNA variants may be selectively degraded to produce single-stranded DNA (ssDNA) variants. The ssDNA variants may be hybridized to ssDNA intermediaries, e.g., uracilated circular ssDNA intermediaries, to form heteroduplex DNA, which may be transformed into cells, such as E. coli cells, yielding a library of variants. This method eliminates the inefficient sub-cloning steps and the need for costly primer sets required by many prior methods.