Claims
- 1. A method for the selection or screening of an antibody fragment library for binding to a (poly)peptide/protein in bacterial host cells comprising the step of employing a Protein Fragment Complementation Assay.
- 2. The method of claim 1, wherein said Protein Fragment Complementation Assay is a DHFR Protein Fragment Complementation Assay.
- 3. The method of claim 1, wherein each of the host cells used in said assay comprises:
(a) a first nucleic acid sequence encoding a first fusion protein comprising a first part of a reporter molecule fused to an antibody fragment member of said library, optionally via a first linker, and (b) a second nucleic acid sequence encoding a second fusion protein comprising a second part of said reporter molecule fused to said (poly)peptide/protein optionally via a second linker, and (c) wherein said first and said second parts of said reporter molecule are able to restore reporter molecule activity by reassociation of said first and said second parts through binding of said antibody fragment to said (poly)peptide/protein.
- 4. The method of claim 3, wherein said reporter molecule is DHFR.
- 5. The method according to claim 4, wherein said first part of said DHFR molecule is mDHFR I, and wherein said second part is mDHFR II.
- 6. A method for the selection or screening of an antibody fragment library for binding to a (poly)peptide/protein in bacterial host cells, comprising the steps of:
(a) expressing in said host cell
(i) a first nucleic acid sequence encoding a first fusion protein comprising a first part of a reporter molecule fused to an antibody fragment member of the library, optionally via a first linker, and (ii) a second nucleic acid sequence encoding a second fusion protein comprising a second part of a reporter molecule fused to the (poly)peptide/protein, optionally via a second linker; (b) allowing said first and said second fusion proteins to contact each other, and (c) screening or selecting for bacterial host cells containing restored reporter molecule activity.
- 7. The method according to claim 6, wherein said first part of said reporter molecule is fused to said antibody fragment member via a first linker.
- 8. The method according to claim 7, wherein said second part of said reporter molecule is fused to said (poly)peptide/protein member via a second linker.
- 9. The method according to claim 8, wherein said reporter molecule is a DHFR molecule.
- 10. The method according to claim 3, wherein said first linker comprises the sequence (Gly4Ser)n, wherein n is between 0 and 6.
- 11. The method according to claim 10, wherein n=4.
- 12. The method according to claim 3, wherein said second linker comprises the sequence (Gly4Ser)n, wherein n is between 0 and 6.
- 13. The method according to claim 12, wherein n=2.
- 14. The method according to claim 3, wherein said first linker comprises the sequence (Gly4Ser)4 and wherein said second linker comprises the sequence (Gly4Ser)2.
- 15. The method according to claim 2, wherein said antibody fragment library comprises scFv fragments.
- 16. A nucleic acid sequence encoding a fusion protein comprising:
(i) a first part of a reporter molecule, (ii) optionally a linker, and (iii) an antibody or a functional antibody fragment.
- 17. The nucleic acid sequence according to claim 16, wherein said fusion protein comprises a linker.
- 18. A nucleic acid molecule according to claim 17, wherein said reporter molecule is a cleavable enzyme.
- 19. A nucleic acid molecule according to claim 18, wherein said reporter molecule is a DHFR molecule.
- 20. A nucleic acid according to claim 19, wherein said first part of a DHFR molecule is mDHFR I.
- 21. A nucleic acid according to claim 19, wherein said linker comprises the sequence (Gly4Ser)n, wherein n is between 0 and 6.
- 22. A nucleic acid according to claim 21, wherein n=4.
- 23. A vector comprising the nucleic acid sequence according to claim 16.
- 24. A bacterial host cell comprising a nucleic acid sequence according to claim 16.
- 25. A bacterial host cell comprising a vector according to claim 23.
- 26. A fusion polypeptide comprising
a. a reporter molecule fragment moiety, b. an antibody or functional antibody fragment moiety, and c. optionally a linker positioned between said moieties.
- 27. A fusion protein according to claim 26, comprising a linker positioned between said reporter molecule fragment moiety and said antibody or said functional antibody fragment moiety.
- 28. A composition of at least two polypeptide chains comprising
a. a first fusion polypeptide comprising
i. a first reporter molecule fragment moiety, ii. an antibody or functional antibody fragment moiety, and iii. optionally a first linker positioned between said moieties; and b. a second fusion polypeptide comprising
i. a second reporter molecule fragment moiety, ii. an antigen moiety, and iii. optionally a second linker positioned between said moieties, wherein said first and second fusion polypeptides interact via said antibody moiety and said antigen moiety, and wherein said first and second reporter molecule fragment moieties associate to form a functional reporter molecule upon association of said antibody moiety and said antigen moiety.
- 29. A composition according to claim 28, wherein said first fusion polypeptide comprises a first linker positioned between said first reporter molecule fragment moiety and said antibody or functional antibody fragment moiety.
- 30. A composition according to claim 29, wherein said second fusion polypeptide comprises a second linker positioned between said second reporter molecule fragment moiety and said antigen moiety.
- 31. A kit comprising
(a) a plurality of nucleic acid sequences capable of encoding a fusion protein, each of said fusion proteins comprising:
(i) a first part of a reporter molecule, (ii) optionally a linker, and (iii) an antibody or functional antibody fragment; and (b) instructions for using said nucleic acid sequences to isolate an antigen of interest.
- 32. A kit according to claim 31, wherein each of said fusion proteins comprises a linker.
- 33. A kit according to claim 32, wherein said antibody fragments represent an antibody library.
- 34. A kit according to claim 32, wherein said reporter molecule is a DHFR molecule.
- 35. A method for adapting members of a library of nucleic acids for use in a protein complementation assay, comprising the step of:
modifying the members of said library to encode a fragment of a reporter molecule, wherein said members encode an antibody or functional fragment thereof.
- 36. A method according to claim 35, wherein said reporter molecule is a DHFR molecule.
- 37. A method according to claim 36, wherein said fragment of DHFR is mDHFR I or mDHFR II.
- 38. The method according to claim 8, wherein the reporter molecule is a beta-lactamase molecule.
- 39. A kit according to claim 32, wherein said reporter molecule is a beta-lactamase molecule.
- 40. A method according to claim 35, wherein said reporter molecule is a β-lactamase molecule.
Parent Case Info
[0001] The present application claims the benefit, under 35 U.S.C. § 119(e), to U.S. provisional patent application Ser. No. 60/329,763, which is hereby incorporated by reference in its entirety.
Provisional Applications (1)
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Number |
Date |
Country |
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60329763 |
Oct 2001 |
US |