Fusions of scaffold proteins with random peptide libraries

• US 6,548,249 B1
• Filed: 07/27/2000
• Issued: 04/15/2003
• Est. Priority Date: 10/08/1998
• Status: Expired due to Term

• Current Assignee: Rigel Pharmaceuticals, Inc.
• Original Assignee: Rigel Pharmaceuticals, Inc.
• Inventors: Anderson, David, Peelle, Beau Robert, Bogenberger, Jakob Maria
• Primary Examiner(s): Brusca, John S.
• Application Number:
  US09/626,581
• Time in Patent Office: 992 Days
• Field of Search: 435/6, 435/320.1, 536/23.4, 536/23.5
• US Class Current: 506/10
• CPC Class Codes: C07K 14/43595 from coelenteratae, e.g. medusae
• CPC Class Codes: C07K 2319/00 Fusion polypeptide
• CPC Class Codes: C07K 2319/42 containing a HA(hemagglutinin)-tag
• CPC Class Codes: C07K 2319/60 containing spectroscopic/fluorescent detection, e.g. green fluorescent protein [GFP]
• CPC Class Codes: C07K 2319/70 containing domain for protein-protein interaction
• CPC Class Codes: C12N 15/1034 Isolating an individual clone by screening libraries
• CPC Class Codes: C12N 15/1044 Preparation or screening of libraries displayed on scaffold proteins
• CPC Class Codes: C12N 15/1086 Preparation or screening of expression libraries, e.g. reporter assays
• CPC Class Codes: C12N 15/62 DNA sequences coding for fusion proteins
• CPC Class Codes: C12Q 1/6897 involving reporter genes operably linked to promoters

Abstract

The invention relates to the use of scaffold proteins, particularly green fluorescent protein (GFP), in fusion constructs with random and defined peptides and peptide libraries, to increase the cellular expression levels, decrease the cellular catabolism, increase the conformational stability relative to linear peptides, and to increase the steady state concentrations of the random peptides and random peptide library members expressed in cells for the purpose of detecting the presence of the peptides and screening random peptide libraries. N-terminal, C-terminal, dual N- and C-terminal and one or more internal fusions are all contemplated. Novel fusions utilizing self-binding peptides to create a conformationally stabilized fusion domain are also contemplated.

Claims

1. A method of screening for bioactive peptides conferring a particular phenotype comprising:

a) providing cells containing a fusion nucleic acid comprising;

i) a first nucleic acid encoding a Renilla GFP scaffold protein;

ii) a second nucleic acid encoding a different random peptide fused to the N-terminus of said scaffold protein; and

iii) a third nucleic acid encoding a flexible linker between said scaffold and said random peptide; and

b) screening said cells for an altered phenotype.

5. A method according to claim 1, 2 or 3, wherein said linker replaces at least one amino acid of said scaffold protein.

6. A method according to claim 1, 2 or 3, wherein said linker comprises -(gly)n-, wherein n≧

2.

7. A method according to claim 1, 2 or 3, wherein said Renilla GFP is wild-type.

8. A method according to claim 1, 2 or 3, wherein said Renilla GFP is a variant GFP.

9. A method according to claim 1, 2 or 3 wherein said cells are mammalian cells.

10. A method according to claim 1, 2 or 3 wherein said providing is accomplished by transfecting said cells with a retroviral vector comprising said fusion nucleic acid.

11. The method according to claim 1, 2 or 3, wherein said linker comprises (GS)_n, wherein n≧

3.

12. The method according to claim 1, 2 or 3, wherein said linker comprises (GSGGS)_n, wherein n≧

1.

13. The method according to claim 1, 2 or 3, wherein said linker comprises (GGGS)_n, wherein n≧

1.

14. The method according to claim 1, 2 or 3, wherein said linker comprises GQGGG.

15. The method according to claim 1, 2 or 3, wherein said linker comprises GQAGGGG.

16. The method according to claim 1, 2, or 3, wherein said phenotype is modulation of an immune response.

17. The method according to claim 1, 2, or 3 wherein said cells comprise T-cells and said phenotype is a T-cell response.

18. The method of claim 17, wherein said T-cell response is proliferation in response to antigen presentation.

19. The method according to claim 1, 2, or 3, wherein said cells comprise B cells and said phenotype is a interaction with a specific immunoglobulin.

20. The method according to claim 1, 2, or 3 wherein said phenotype is cytokine production.

21. The method according to claim 20, wherein said cells are macrophages.

22. The method according to claim 20, wherein said cells are monocytes.

23. The method according to claim 1, 2, or 3 wherein said cells are mononuclear leukocytes and said phenotype is antigen processing.

24. The method according to claim 1, 2, or 3 wherein said phenotype is release of inflammatory mediators.

25. The method of claim 24 wherein said cells comprise mononuclear leukocytes.

26. The method of claim 24 wherein said cells comprise mast cells.

27. The method of claim 24 wherein said cells comprise eosinophils.

2. A method of screening for bioactive peptides conferring a particular phenotype comprising:

a) providing cells containing a fusion nucleic acid comprising;

i) a first nucleic acid encoding a Renilla GFP scaffold protein;

ii) a second nucleic acid encoding a different random peptide fused to the C-terminus of said scaffold protein; and

iii) a third nucleic acid encoding a flexible linker between said scaffold and said random peptide; and

b) screening said cells for an altered phenotype.

3. A method of screening for bioactive peptides conferring a particular phenotype comprising:

a) providing cells containing a fusion nucleic acid comprising;

i) a first nucleic acid encoding a Renilla GFP scaffold protein;

ii) a second nucleic acid encoding a different random peptide wherein said second nucleic acid is inserted into said first nucleic acid; and

iii) a third nucleic acid encoding a first flexible linker between said scaffold and said random peptide; and

b) screening said cells for an altered phenotype.

4. A method according to claim 3 further comprising a second flexible linker between the other end of said random peptide and said scaffold protein.

28. A method of screening for bioactive peptides conferring a particular phenotype comprising:

a) providing cells containing a fusion nucleic acid comprising;

i) a first nucleic acid encoding a Renilla GFP scaffold protein;

ii) a second nucleic acid encoding a different random peptide fused to the N-terminus of said scaffold protein; and

iii) a third nucleic acid encoding a linker between said scaffold and said random peptide, wherein said linker comprises -(gly)n-, wherein n≧

2; and

b) screening said cells for an altered phenotype.

29. A method of screening for bioactive peptides conferring a particular phenotype comprising:

a) providing cells containing a fusion nucleic acid comprising;

i) a first nucleic acid encoding a Renilla GFP scaffold protein;

ii) a second nucleic acid encoding a different random peptide fused to the C-terminus of said scaffold protein; and

iii) a third nucleic acid encoding a linker between said scaffold and said random peptide, wherein said linker comprises -(gly)n-, wherein n≧

2; and

b) screening said cells for an altered phenotype.

30. A method of screening for bioactive peptides conferring a particular phenotype comprising:

a) providing cells containing a fusion nucleic acid comprising;

i) a first nucleic acid encoding a Renilla GFP scaffold protein;

ii) a second nucleic acid encoding a different random peptide wherein said second nucleic acid is inserted into said first nucleic acid; and

iii) a third nucleic acid encoding a linker between said scaffold and said random peptide, wherein said linker comprises -(gly)n-, wherein n≧

2; and

b) screening said cells for an altered phenotype.

31. A method of screening for bioactive peptides conferring a particular phenotype comprising:

a) providing cells containing a fusion nucleic acid comprising;

i) a first nucleic acid encoding a Renilla GFP scaffold protein;

ii) a second nucleic acid encoding a different random peptide fused to the N-terminus of said scaffold protein; and

iii) a third nucleic acid encoding a linker between said scaffold and said random peptide, wherein said linker replaces at least one amino acid of said scaffold protein; and

b) screening said cells for an altered phenotype.

32. A method of screening for bioactive peptides conferring a particular phenotype comprising:

a) providing cells containing a fusion nucleic acid comprising;

i) a first nucleic acid encoding a Renilla GFP scaffold protein;

ii) a second nucleic acid encoding a different random peptide fused to the C-terminus of said scaffold protein; and

iii) a third nucleic acid encoding a linker between said scaffold and said random peptide, wherein said linker replaces at least one amino acid of said scaffold protein; and

b) screening said cells for an altered phenotype.

33. A method of screening for bioactive peptides conferring a particular phenotype comprising:

a) providing cells containing a fusion nucleic acid comprising;

i) a first nucleic acid encoding a Renilla GFP scaffold protein;

ii) a second nucleic acid encoding a different random peptide wherein said second nucleic acid is inserted into said first nucleic acid; and

iii) a third nucleic acid encoding a linker between said scaffold and said random peptide, wherein said linker replaces at least one amino acid of said scaffold protein; and