Peptide library and screening systems

Claim 1

A method for obtaining a bacteriophage encoding a substrate cleaved by a preselected proteolytic enzyme, comprising: (a) transforming host cells with at least 10 6 different bacteriophage expression vectors, wherein each of said vectors encodes a fusion protein composed of a random peptide fused to a known ligand for a receptor, which known ligand is in turn fused to a coat protein of a filamentous bacteriophage wherein said vectors differ from each other with respect to said random peptides of said fusion protein encoded by said vector and said ligand will not bind said receptor when said ligand is fused to said random peptide: (b) cultivating said transformed host cells under conditions suitable for expression and assembly of bacteriophage; (c) incubating said bacteriophage with said preselected proteolytic enzyme under conditions such that a random peptide displayed by said bacteriophage and cleavable by said proteolytic enzyme is cleaved; (d) contacting said bacteriophage from step (c) to said receptor that binds said known ligand under conditions conducive to specific receptor-ligand binding; and (e) separating receptor-bound bacteriophage from unbound bacteriophage; and thereby obtaining said bacteriophage encoding said substrate for said preselected proteolytic enzyme.

Claim 2

A method for obtaining a bacteriophage encoding a substrate cleaved by a preselected proteolytic enzyme, comprising: (a) transforming host cells with at least 10 6 different bacteriophage expression vectors, wherein each of said vectors encodes a fusion protein composed of a random peptide fused to a known ligand for a receptor, which known ligand is in turn fused to a coat protein of a filamentous bacteriophage wherein said vectors differ from each other with respect to said random peptides of said fusion protein encoded by said vector and said ligand will not bind said receptor when said ligand is fused to said random peptide: (b) cultivating said transformed host cells under conditions suitable for expression and assembly of bacteriophage; (c) incubating said bacteriophage with said preselected proteolytic enzyme under conditions such that a random peptide displayed by said bacteriophage and cleavable by said proteolytic enzyme is cleaved; (d) contacting said bacteriophage from step (c) to said receptor that binds said known ligand under conditions conducive to specific receptor-ligand binding; and (e) separating receptor-bound bacteriophage from unbound bacteriophage; and thereby obtaining said bacteriophage encoding said substrate for said preselected proteolytic enzyme.

Claim 3

A method for obtaining a bacteriophage encoding a substrate cleaved by a preselected proteolytic enzyme, comprising; (a) transforming host cells with at least 10 6 different bacteriophage expression vectors, wherein each of said vectors encodes a fusion protein composed of a known ligand for a receptor fused to a random peptide, which random peptide is in turn fused to a coat protein of a filamentous bacteriophage, and wherein said vectors differ from each other with respect to said random peptide of said fusion protein encoded by said vector; (b) cultivating said transformed host cells under conditions suitable for expression and assembly of bacteriophage; (c) contacting said bacteriophage from step (b) to said receptor that binds said known ligand under conditions conducive to specific receptor-ligand binding; (d) incubating said bacteriophage bound to said receptor with said preselected proteolytic enzyme under conditions such that a random peptide cleavable by said proteolytic enzyme is cleaved; (e) separating bacteriophage which are released when said random peptide is cleaved and thereby obtaining said bacteriophage encoding said substrate for said preselected enzyme.

Claim 4

A method for obtaining a bacteriophage encoding a substrate cleaved by a preselected proteolytic enzyme, comprising; (a) transforming host cells with at least 10 6 different bacteriophage expression vectors, wherein each of said vectors encodes a fusion protein composed of a known ligand for a receptor fused to a random peptide, which random peptide is in turn fused to a coat protein of a filamentous bacteriophage, and wherein said vectors differ from each other with respect to said random peptide of said fusion protein encoded by said vector; (b) cultivating said transformed host cells under conditions suitable for expression and assembly of bacteriophage; (c) contacting said bacteriophage from step (b) to said receptor that binds said known ligand under conditions conducive to specific receptor-ligand binding; (d) incubating said bacteriophage bound to said receptor with said preselected proteolytic enzyme under conditions such that a random peptide cleavable by said proteolytic enzyme is cleaved; (e) separating bacteriophage which are released when said random peptide is cleaved and thereby obtaining said bacteriophage encoding said substrate for said preselected enzyme.

Claim 5

A method for identifying a peptide which binds to a preselected receptor and measuring a dissociation rate for the binding of said peptide to said receptor, comprising; p1 (a) transforming host cells with at least 10 6 different bacteriophage expression vector wherein each of said vectors encodes a fusion protein composed of a peptide fused to a coat protein of a filamentous bacteriophage, and wherein said vectors differ from each other with respect to the peptide of said fusion protein encoded by said vector; (b) cultivating said transformed host cells under conditions suitable for expression and assembly of bacteriophage; (c) contacting bacteriophage displaying the peptide to the preselected receptor under conditions conducive to specific peptide-receptor binding; (d) selecting bacteriophage which bind to the receptor; (e) separating bound bacteriophage selected in step (d) into individual isolates; (f) binding each isolate separated in step (e) to a labeled monovalent receptor; and (g) measuring over time how much receptor binds each isolate in the presence and absence of a known ligand for said receptor to determine a dissociation rate for the binding of said peptide to said receptor.

Claim 6

A method for identifying a peptide which binds to a preselected receptor and measuring a dissociation rate for the binding of said peptide to said receptor, comprising; p1 (a) transforming host cells with at least 10 6 different bacteriophage expression vector wherein each of said vectors encodes a fusion protein composed of a peptide fused to a coat protein of a filamentous bacteriophage, and wherein said vectors differ from each other with respect to the peptide of said fusion protein encoded by said vector; (b) cultivating said transformed host cells under conditions suitable for expression and assembly of bacteriophage; (c) contacting bacteriophage displaying the peptide to the preselected receptor under conditions conducive to specific peptide-receptor binding; (d) selecting bacteriophage which bind to the receptor; (e) separating bound bacteriophage selected in step (d) into individual isolates; (f) binding each isolate separated in step (e) to a labeled monovalent receptor; and (g) measuring over time how much receptor binds each isolate in the presence and absence of a known ligand for said receptor to determine a dissociation rate for the binding of said peptide to said receptor.

Claim 7

The method of any one of claims 1, 3 or 2, where the receptor is bound to a solid phase and the selected bacteriophage are separated from the culture.

Claim 8

The method of any one of claims 1, 3 or 2, wherein the filamentous bacteriophage is f1, fd, or M13.

Claim 11

The method of any one of claims 1, 3 or 2, wherein each of the 10 6 different bacteriophage expression vectors comprises a series of codons encoding a random collection of amino acids of which said peptide is comprised.

Claim 13

The method of any one of claims 1, 3 or 2, wherein the host cells are transformed by electroporation.

Claim 14

The method of any one of claims 1, 3 or 2, wherein the oligonucleotide library comprises at least about 10 8 different expression vectors.

Claim 15

The method of any one of claims 1, 3 or 2, wherein the coat protein of the bacteriophage is a preprotein which is processed by the host cell to display the peptide so that an N-terminal amino acid of the fusion protein is an N-terminal amino acid of said peptide.