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1. WO2007010525 - METHODS AND COMPOSITIONS FOR IDENTIFYING A PEPTIDE HAVING AN INTERMOLECULAR INTERACTION WITH A TARGET OF INTEREST

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[ EN ]

WHAT IS CLAIMED IS:

1. A recombinant vims peptide library wherein each recombinant virus comprises:

a. at least one protein or protein fragment, comprising segments which are involved in viral attachment to, infection of, or a combination thereof of a host cell;

b. a peptide or polypeptide, which differs by at least one amino acid from another peptide or polypeptide in said library; and

c. a modified cleavage site proximal to said peptide and to said segments of said protein,

wherein said cleavage site is modified such that a compound mediating cleavage has a reduced binding affinity for said site, as compared to a non-modified cleavage site.

2. The recombinant vims library of claim 1, wherein said peptide or polypeptide is involved in at least one intermolecular interaction.

3. The recombinant vims library of claim 1, wherein said peptide or polypeptide is an agonist, antagonist, antigen, enzyme, enzyme activator, enzyme inhibitor, enzyme substrate, hormone, regulatory protein, cytoskeletal protein, toxin, or a functional fragment thereof.

4. The recombinant virus library of claim 1, wherein said cleavage site is located between:

a. a protein or protein fragment, comprising segments which are involved in viral attachment, infection, or a combination thereof; and

b. a protein or protein fragment, comprising segments that serve as an anchor.

5. The recombinant virus library of claim 1, wherein said reduced binding affinity is reflected as a modified off-rate, a modified on-rate, or an altered binding enthalpy.

6. A plurality of oligonucleotide vectors of the recombinant virus library of claim 1.

7. A plurality of cells comprising the recombinant virus library of claim 1.

8. A recombinant virus comprising:

a. at least one protein or protein fragment, comprising segments which are involved in viral attachment to, infection of, or a combination thereof of a host cell;

b. a peptide or polypeptide involved in an intermolecular interaction; and

c. a modified cleavage site proximal to said peptide and to said segments of said protein,

wherein said cleavage site is modified such that a compound mediating cleavage has a reduced binding affinity for said site, as compared to a non-modified cleavage site.

9. The recombinant virus of claim 8, wherein said peptide or polypeptide is an agonist, antagonist, antigen, enzyme, enzyme activator, enzyme inhibitor, enzyme substrate, hormone, regulatory protein, cytoskeletal protein, toxin, or a functional fragment thereof.

10. The recombinant virus of claim 8, wherein said cleavage site is located between:

a. a protein or protein fragment, comprising segments which are involved in viral attachment, infection, or a combination thereof; and

b. a protein or protein fragment, comprising segments that serve as an anchor.

11. The recombinant virus of claim 8. wherein said reduced binding affinity is reflected as a modified off-rate, a modified on-rate, or an altered binding enthalpy.

12. A oligonucleotide vector of the recombinant virus library of claim 8.

13. A cell comprising the recombinant virus of claim 8.

14. A target of interest complex comprising:

a. a protease;

b. a flexible linker attached to said protease; and

c. a target of interest attached to said flexible linker,

wherein said target of interest participates in an intermolecular interaction.

15. The complex of claim 14, wherein the target of interest is an agonist, antagonist, receptor, antibody, antigen, enzyme, enzyme activator, enzyme inhibitor, enzyme substrate, hormone, regulatory protein, cytoskeletal protein, toxin, synthetic or physiological polymer, small organic molecule, DNA, RNA, oligonucleotide, transcriptional activator or repressor, translational activator or repressor, or a functional fragment thereof.

16. The target of interest of claim 14, wherein said target of interest has one or more intermolecular interactions with a cognate ligand, antigen, enzyme substrate, enzyme, regulatory protein, or cytoskeletal protein expressed on the surface of a recombinant virus.

17. The complex of claim 14, wherein the flexible linker is at least 3 amino acids in length.

18. A method of identifying a peptide or polypeptide, which has an intermolecular interaction with a target of interest comprising the steps of:

a. contacting the recombinant virus library of claim 1 with a plurality of complexes of claim 14;

b. contacting the library of (a) with a plurality of cells;

c. isolating viruses in (b) which have not infected said cells;

d. providing infectious clones of isolated viruses of step (c) by amplifying and expressing the genomes of said isolated viruses;

e. repeating steps (a - d);

f. identifying peptides expressed by the viruses in step (c);

whereby intermolecular interactions between said target of interest and a peptide expressed by said recombinant virus result in said protease being in close proximity to said cleavage site, resulting in cleavage of said protein or protein fragment, comprising segments which are involved in viral attachment to, infection of, or a combination thereof of cells, and prevention of entry of a virus that had comprised a peptide involved in intermolecular interactions into said cells.

19. The method of claim 18, wherein the recombinant virus library is produced in a phage.

20. The method of claim 18, wherein the recombinant virus library is produced in M13 bacteriophage.

21. The method of claim 18, wherein recombinant viruses of said library comprise peptides with mutations that abrogate binding to said target of interest.

22. The method of claim 18, wherein said peptide or polypeptide is used for epitope mapping of the target of interest.

23. The method of claim 18, wherein said peptide or polypeptide is an agonist, antagonist, antigen, enzyme, enzyme activator, enzyme inhibitor, enzyme substrate, hormone, regulatory protein, cytoskeletal protein, toxin, transcriptional activator or repressor, translational activator or repressor, or a functional fragment thereof.

24. The method of claim 18, wherein said target of interest is an agonist, antagonist, receptor, antibody, antigen, enzyme, enzyme activator, enzyme inhibitor, enzyme substrate, hormone, regulatory protein, cytoskeletal protein, toxin, synthetic or physiological polymer, small organic molecule, DNA, RNA, oligonucleotide, transcriptional activator or repressor, translational activator or repressor, or a functional fragment thereof.

25. The method of claim 18, wherein said target of interest is not bound to a solid support.

26. The method of claim 18, wherein the viruses in step (f) which are isolated are attached to the surface, but have not entered, said cells.

27. A kit for identifying a peptide or polypeptide involved in an intermolecular interaction with a target of interest comprising:

a. a recombinant virus peptide library of claim 1 ;

b. a target of interest attached to a protease via a flexible linker; and

c. cells that are susceptible to viral attachment, infection or a combination thereof.

28. The kit of claim 27, wherein the recombinant virus library is produced in a phage.

29. The kit of claim 27, wherein the recombinant virus library is produced in Ml 3 bacteriophage.

30. The kit of claim 27, wherein recombinant viruses of said library comprise peptides with mutations that abrogate binding to said target of interest.

31. The kit of claim 27, wherein said peptide or polypeptide is used for epitope mapping of the target of interest.

32. The kit of claim 27, wherein said peptide or polypeptide is an agonist, antagonist, antigen, enzyme, enzyme activator, enzyme inhibitor, enzyme substrate, hormone, regulatory protein, cytoskeletal protein, toxin, transcriptional activator or repressor, translational activator or repressor, or a functional fragment thereof.

33. The kit of claim 27, wherein said target of interest is an agonist, antagonist, receptor, antibody, antigen, enzyme, enzyme activator, enzyme inhibitor, enzyme substrate, hormone, regulatory protein, cytoskeletal protein, toxin, synthetic or physiological polymer, small organic molecule, DNA, RNA, oligonucleotide, transcriptional activator or repressor, translational activator or repressor, or a functional fragment thereof.

34. The kit of claim 27, wherein said target of interest is not bound to a solid support.

35. A method of identifying an agonistic or antagonistic feature of a peptide or polypeptide, which has an intermolecular interaction with a receptor of interest comprising the steps of:

a. contacting the recombinant virus library of claim 1 with a plurality of complexes of claim 14, wherein the target of interest is a receptor;

b. contacting said recombinant virus library of step (a) with cells;

c. isolating viruses in (b) which have not infected said cells;

d. providing infectious clones of first isolated viruses of (c) by amplifying and expressing the genomes of said first isolated viruses;

e. contacting infectious clones of viruses of step (c) with the receptor of interest, wherein said receptor is not attached to a protease;

f. contacting the viruses of step (e) with a protein attached to a protease via a flexible linker, wherein said protein is involved in the downstream signal transduction pathway of said receptor of interest;

g. contacting the viruses of step (f) with cells;

h. separating viruses in (g) that have not infected said cells from viruses which have infected cells;

i. providing infectious clones of second isolated viruses of step (h) by amplifying and expressing the genomes of said second isolated viruses;

j. repeating steps (a) - (i); and k. identifying peptides expressed by the viruses in step (h),

whereby viruses which have not infected said cells in (h) express a peptide which has agonistic activity for said receptor of interest, and viruses which have infected said cells in (h) express a peptide which has antagonistic activity for said receptor.

36. A method of identifying a peptide or polypeptide that inhibits an enzyme of interest comprising the steps of:

a. contacting the recombinant vims library of claim 1 with a plurality of complexes comprising of claim 14, wherein the target of interest is an enzyme;

b. contacting said recombinant virus library of step (a) with cells;

c. isolating viruses in (b) which have not infected said cells;

d. providing infectious clones of first isolated viruses of (c) by amplifying and expressing the genomes of said first isolated viruses;

e. contacting infectious clones of viruses of step (c) with the enzyme of interest, wherein said enzyme is not attached to a protease;

f. contacting the viruses of step (e) with a substrate of the enzyme attached to a protease via a flexible linker;

g. contacting the viruses of step (f) with cells;

h. separating viruses in (g) which have not infected said cells from viruses which have infected cells;

i. providing infectious clones of second isolated viruses of step (i) by amplifying and expressing the genomes of said second isolated viruses;

j. repeating steps (a) - (i); and

k. identifying peptides expressed by the viruses in step (h),

whereby viruses which have not infected said cells in (h) express a peptide which does not affect said enzyme of interest, and viruses which have infected said cells in (h) express a peptide which inhibits said enzyme.

37. The methods of claims 35 and 36, wheicm the recombinant virus library is produced in a phage.

38. The methods of claims 35 and 36, wherein the recombinant virus library is produced in Ml 3 bacteriophage.

39. The methods of claims 35 and 36, wherein recombinant viruses of said library comprise peptides with mutations that abrogate binding to said receptor or enzyme of interest.

40. The methods of claims 35 and 36, wherein said receptor or enzyme of interest is not bound to a solid support.

41. The methods of claims 35 and 36, wherein isolated viruses of steps (c) and (h) are attached to the surface of said cells.