Peptide library and screening method

Claim 1

A method of isolating a DNA binding protein comprising: (a) providing a recombinant DNA vector comprising a coding sequence for a peptide having a specific affinity for a receptor in frame with a DNA segment encoding a linker; (b) inserting a library of oligonucleotides encoding different potential DNA binding proteins into multiple copies of the recombinant DNA vector in frame with the peptide coding sequence and the linker to form a library of different vectors encoding different fusion proteins, each comprising a potential DNA binding protein, a linker and the peptide, the fusion proteins differing in the potential DNA binding proteins; (c) transforming host cells with the library of different vectors to form transformed host cells; (d) culturing the transformed host cells under conditions suitable for expression of the fusion proteins, whereby, if a fusion protein comprises a potential DNA binding protein with affinity for a vector encoding the fusion protein, the fusion protein binds to the vector to form a complex; (e) lysing the transformed host cells under conditions such that complexes formed in (d) remain associated; (f) contacting the complexes with a receptor under conditions conducive to specific binding of the peptide to the receptor; (g) isolating complexes bound to the receptor, the complexes containing vectors encoding DNA binding proteins.

Claim 2

A method of isolating a DNA binding protein comprising: (a) providing a recombinant DNA vector comprising a coding sequence for a peptide having a specific affinity for a receptor in frame with a DNA segment encoding a linker; (b) inserting a library of oligonucleotides encoding different potential DNA binding proteins into multiple copies of the recombinant DNA vector in frame with the peptide coding sequence and the linker to form a library of different vectors encoding different fusion proteins, each comprising a potential DNA binding protein, a linker and the peptide, the fusion proteins differing in the potential DNA binding proteins; (c) transforming host cells with the library of different vectors to form transformed host cells; (d) culturing the transformed host cells under conditions suitable for expression of the fusion proteins, whereby, if a fusion protein comprises a potential DNA binding protein with affinity for a vector encoding the fusion protein, the fusion protein binds to the vector to form a complex; (e) lysing the transformed host cells under conditions such that complexes formed in (d) remain associated; (f) contacting the complexes with a receptor under conditions conducive to specific binding of the peptide to the receptor; (g) isolating complexes bound to the receptor, the complexes containing vectors encoding DNA binding proteins.

Claim 11

A method of constructing a random peptide library, comprising: (a) providing a recombinant DNA vector that encodes a DNA binding protein other than a phage coat protein; (b) inserting into the coding sequence of the DNA binding protein a coding sequence for a random peptide such that the resulting vectors encode fusion proteins, each of which comprises the DNA binding protein and a random peptide; (c) transforming host cells with the vectors; and (d) culturing the transformed host cells under conditions suitable for expression of the fusion proteins, wherein the fusion proteins bind via the DNA binding protein to the vector to form complexes; (e) contacting the complexes formed in step (d) with a receptor, whereby complexes having a random peptide with specific affinity for a receptor are enriched by affinity purification to the receptor from complexes lacking a random peptide with specific affinity for the receptor; (f) determining the DNA sequence encoding the random peptide of a vector in complex enriched by affinity purification in step (e); (g) preparing a plurality of oligonucleotides having sequences that are variants of the sequence determined in step (f); h) repeating steps (b)-(e) with the plurality of oligonucleotides being inserted into the DNA coding sequence of the DNA binding protein in step (b) in place of the sequence encoding the random peptide.

Claim 12

A method of constructing a random peptide library, comprising: (a) providing a recombinant DNA vector that encodes a DNA binding protein other than a phage coat protein; (b) inserting into the coding sequence of the DNA binding protein a coding sequence for a random peptide such that the resulting vectors encode fusion proteins, each of which comprises the DNA binding protein and a random peptide; (c) transforming host cells with the vectors; and (d) culturing the transformed host cells under conditions suitable for expression of the fusion proteins, wherein the fusion proteins bind via the DNA binding protein to the vector to form complexes; (e) contacting the complexes formed in step (d) with a receptor, whereby complexes having a random peptide with specific affinity for a receptor are enriched by affinity purification to the receptor from complexes lacking a random peptide with specific affinity for the receptor; (f) determining the DNA sequence encoding the random peptide of a vector in complex enriched by affinity purification in step (e); (g) preparing a plurality of oligonucleotides having sequences that are variants of the sequence determined in step (f); h) repeating steps (b)-(e) with the plurality of oligonucleotides being inserted into the DNA coding sequence of the DNA binding protein in step (b) in place of the sequence encoding the random peptide.

Claim 14

A method for screening a random peptide library for a peptide with specific affinity for a receptor, comprising: (a) providing a peptide library wherein each member is a host cell transformed with a recombinant DNA vector that encodes a fusion protein comprising a DNA binding protein, a coding sequence for a random peptide, and a linker peptide which links the DNA binding protein and the random peptide, wherein members differ from other members with respect to the sequence of the random peptide, wherein the fusion proteins can bind via the DNA binding protein to the vector to form complexes having sufficient stability that complexes having a random peptide with a specific affinity for a receptor can be enriched by affinity purification to the receptor from complexes lacking a random peptide with a specific affinity for the receptor; (b) lysing the cells transformed with the random peptide library under conditions such that the fusion protein remains bound to the vector that encodes the fusion protein; (c) contacting the fusion proteins of the random peptide library with a receptor under conditions conducive to specific peptide-receptor binding; and (d) isolating the vector that encodes a random peptide that binds to said receptor.

Claim 15

A method for screening a random peptide library for a peptide with specific affinity for a receptor, comprising: (a) providing a peptide library wherein each member is a host cell transformed with a recombinant DNA vector that encodes a fusion protein comprising a DNA binding protein, a coding sequence for a random peptide, and a linker peptide which links the DNA binding protein and the random peptide, wherein members differ from other members with respect to the sequence of the random peptide, wherein the fusion proteins can bind via the DNA binding protein to the vector to form complexes having sufficient stability that complexes having a random peptide with a specific affinity for a receptor can be enriched by affinity purification to the receptor from complexes lacking a random peptide with a specific affinity for the receptor; (b) lysing the cells transformed with the random peptide library under conditions such that the fusion protein remains bound to the vector that encodes the fusion protein; (c) contacting the fusion proteins of the random peptide library with a receptor under conditions conducive to specific peptide-receptor binding; and (d) isolating the vector that encodes a random peptide that binds to said receptor.

Claim 34

A method of isolating a DNA binding protein comprising: (a) providing a recombinant DNA vector comprising a coding sequence for a peptide having a specific affinity for a receptor and a predetermined DNA sequence for which a DNA binding protein is to be isolated; (b) inserting a library of oligonucleotides encoding different potential DNA binding proteins into multiple copies of the recombinant DNA vector in-frame with the peptide coding sequence to form a library of different vectors encoding different fusion proteins, the fusion proteins differing in the potential DNA binding proteins; (c) transforming host cells with the library of different vectors to form transformed host cells; (d) culturing the transformed host cells under conditions suitable for expression of the fusion proteins, whereby, if a fusion protein comprises a potential DNA binding protein with affinity for a vector encoding the fusion protein, the fusion protein bind, to the predetermined sequence to form a complex; (e) lysing the transformed host cells under conditions such that complexes formed in (d) remain associated; (f) contacting the complexes with a receptor under conditions conducive to specific binding of the peptide to the receptor; (g) isolating complexes bound to the receptor, the complexes containing vectors encoding DNA binding proteins that bind to the predetermined sequence.

Claim 35

A method of isolating a DNA binding protein comprising: (a) providing a recombinant DNA vector comprising a coding sequence for a peptide having a specific affinity for a receptor and a predetermined DNA sequence for which a DNA binding protein is to be isolated; (b) inserting a library of oligonucleotides encoding different potential DNA binding proteins into multiple copies of the recombinant DNA vector in-frame with the peptide coding sequence to form a library of different vectors encoding different fusion proteins, the fusion proteins differing in the potential DNA binding proteins; (c) transforming host cells with the library of different vectors to form transformed host cells; (d) culturing the transformed host cells under conditions suitable for expression of the fusion proteins, whereby, if a fusion protein comprises a potential DNA binding protein with affinity for a vector encoding the fusion protein, the fusion protein bind, to the predetermined sequence to form a complex; (e) lysing the transformed host cells under conditions such that complexes formed in (d) remain associated; (f) contacting the complexes with a receptor under conditions conducive to specific binding of the peptide to the receptor; (g) isolating complexes bound to the receptor, the complexes containing vectors encoding DNA binding proteins that bind to the predetermined sequence.

Claim 40

A method of constructing a DNA binding protein library, comprising: (a) providing a recombinant DNA vector comprising a coding sequence for a peptide having a specific affinity for a receptor; (b) inserting a coding sequence for a potential DNA binding protein into the vector in-frame with the peptide coding sequence such that the resulting vectors encode fusion proteins, each of which comprises a potential DNA binding protein and the peptide with specific affinity for the receptor; (c) transforming host cells with the vectors; (d) culturing the transformed host cells in a solution under conditions suitable for expression of the fusion proteins, whereby, if a fusion protein comprises a potential DNA binding protein with affinity for the vector encoding the fusion protein, the fusion protein binds to the vector to form a complex which has sufficient stability so that the complex can be separated from fusion proteins and vectors that do not form complexes.

Claim 41

A method of constructing a DNA binding protein library, comprising: (a) providing a recombinant DNA vector comprising a coding sequence for a peptide having a specific affinity for a receptor; (b) inserting a coding sequence for a potential DNA binding protein into the vector in-frame with the peptide coding sequence such that the resulting vectors encode fusion proteins, each of which comprises a potential DNA binding protein and the peptide with specific affinity for the receptor; (c) transforming host cells with the vectors; (d) culturing the transformed host cells in a solution under conditions suitable for expression of the fusion proteins, whereby, if a fusion protein comprises a potential DNA binding protein with affinity for the vector encoding the fusion protein, the fusion protein binds to the vector to form a complex which has sufficient stability so that the complex can be separated from fusion proteins and vectors that do not form complexes.