Process for the production of stochastically-generated peptides,polypeptides or proteins having a predetermined property

• US 5,817,483 A
• Filed: 06/05/1995
• Issued: 10/06/1998
• Est. Priority Date: 03/30/1985
• Status: Expired due to Term

• Current Assignee: Stuart Kauffman
• Original Assignee: Stuart Kauffman
• Inventors: Ballivet, Marc, Kauffman, Stuart Alan
• Primary Examiner(s): Robinson, Douglas W.
• Assistant Examiner(s): Wai, Thanda
• Application Number:
  US08/464,141
• Time in Patent Office: 1,219 Days
• Field of Search: 435/69.1, 435/7.1, 435/91.1, 435/172.3, 435/320.1, 536/23.1, 530/300, 530/350
• US Class Current: 435/69.1
• CPC Class Codes: A61K 39/00 Medicinal preparations containing antigens or antibodies materials for immunoassay G01N33/53
• CPC Class Codes: C07K 14/005 from viruses
• CPC Class Codes: C07K 14/485 Epidermal growth factor [EGF], i.e. urogastrone
• CPC Class Codes: C07K 2319/00 Fusion polypeptide
• CPC Class Codes: C12N 15/10 Processes for the isolation, preparation or purification of DNA or RNA chemical preparation of DNA or RNA C07H21/00; preparation of non-structural polynucleotides from microorganisms or with enzymes C12P19/34
• CPC Class Codes: C12N 15/1034 Isolating an individual clone by screening libraries
• CPC Class Codes: C12N 15/1048 SELEX
• CPC Class Codes: C12N 15/1093 General methods of preparing gene libraries, not provided for in other subgroups
• CPC Class Codes: C12N 15/64 General methods for preparing the vector, for introducing it into the cell or for selecting the vector-containing host
• CPC Class Codes: C12N 15/66 General methods for inserting a gene into a vector to form a recombinant vector using cleavage and ligation; Use of non-functional linkers or adaptors, e.g. linkers containing the sequence for a restriction endonuclease
• CPC Class Codes: C12N 15/68 Stabilisation of the vector
• CPC Class Codes: C12N 15/70 Vectors or expression systems specially adapted for E. coli
• CPC Class Codes: C12N 2730/10122 New viral proteins or individual genes, new structural or functional aspects of known viral proteins or genes
• CPC Class Codes: C12N 9/2471 Beta-galactosidase (3.2.1.23), i.e. exo-(1-->4)-beta-D-galactanase
• CPC Class Codes: C12Y 302/01023 Beta-galactosidase (3.2.1.23), i.e. exo-(1-->4)-beta-D-galactanase

Abstract

The present invention is directed to a process for the production of a peptide, polypeptide, or protein having a predetermined property. In accordance with one embodiment, the process begins by producing by way of synthetic polynucleotide coupling, stochastically generated polynucleotide sequences. A library of expression vectors containing such stochastically generated polynucleotide sequences is formed. Next, host cells containing the vectors are cultured so as to produce peptides, polypeptides, or proteins encoded by the stochastically generated polynucleotide sequences. Screening or selection is carried out on such host cells to identify a peptide, polypeptide, or protein produced by the host cells which has the predetermined property. The stochastically generated polynucleotide sequence which encodes the identified peptide, polypeptide, or protein is then isolated and used to produce the peptide, polypeptide, or protein having the predetermined property.

Claims

1. A process for the production of a peptide, polypeptide, or protein having a predetermined property, comprising the steps of:

producing by synthetic polynucleotide coupling, a population of stochastically generated polynucleotide sequences;

forming a library of expression vectors containing said population of stochastically generated polynucleotide sequences;

introducing the vectors into host cells;

culturing the host cells;

carrying out screening or selection on said host cells, to identify a peptide, polypeptide, or protein produced by the host cells having the predetermined property;

isolating a stochastically generated polynucleotide sequence which encodes the identified peptide, polypeptide, or protein;

using the isolated sequence to produce the peptide, polypeptide, or protein having the predetermined property.

2. A process for the production of a peptide, polypeptide, or protein having a predetermined property, comprising the steps of:

producing a population of at least partially stochastic synthetic polynucleotide sequences;

introducing the population of at least partially stochastic polynucleotide sequences into host cells to produce transformed host cells;

cultivating the transformed host cells to produce peptides, polypeptides, or proteins expressed by at least some of the stochastic polynucleotide sequences;

carrying out screening and/or selection methods on said transformed host cells to identify clones producing the peptide, polypeptide, or protein having the predetermined property;

isolating the clones so identified; and

growing the isolated clones in a manner so as to produce the peptide, polypeptide, or protein having the predetermined property.

3. A process for the detection or titration of a ligand using a peptide, polypeptide, or protein having a predetermined property, comprising the steps of:

producing by synthetic polynucleotide coupling, a population of stochastically generated polynucleotide sequences;

forming a library of expression vectors containing said population of stochastically generated polynucleotide sequences;

introducing the vectors into host cells;

culturing the host cells containing the vectors to produce peptides, polypeptides, or proteins encoded by the stochastically generated polynucleotide sequences;

carrying out screening or selection on said host cells, to identify a peptide, polypeptide, or protein produced by the host cells having the predetermined property;

contacting the peptide, polypeptide, or protein with two or more concentrations of a ligand; and

determining the amount of peptide, polypeptide or protein bound at each concentration of ligand.

5. The process according to claim 3 or claim 4, wherein more than one ligand is detected or titrated.

4. A process for the detection or titration of a ligand using a peptide, polypeptide, or protein having a predetermined property, comprising the steps of:

producing a population of at least partially stochastic synthetic polynucleotide sequences;

introducing the population of at least partially stochastic polynucleotide sequences into host cells to produce transformed host cells;

cultivating the transformed host cells to produce peptides, polypeptides, or proteins expressed by at least some of the stochastic polynucleotide sequences;

carrying out screening and/or selection methods on said transformed host cells to identify clones producing the peptide, polypeptide, or protein having the predetermined property;

contacting the peptide, polypeptide, or protein with two or more concentrations of a ligand; and

determining the amount of peptide, polypeptide or protein bound at each concentration of ligand.

6. A method of identifying a peptide, polypeptide or protein having a predetermined property, comprising:

(a) producing a population of peptides, polypeptides or proteins encoded by stochastic polynucleotide sequences;

(b) screening said population of peptides, polypeptides or proteins for said predetermined property under conditions which allow detection of one or more peptides, polypeptides or proteins having said predetermined property.

7. The method of claim 6, wherein step (a) further comprises synthesizing a population of stochastic polynucleotide sequences and translating said population of stochastic polynucleotide sequences to produce said population of peptides, polypeptides or proteins.

8. The method of claim 6, wherein step (a) further comprises synthesizing a population of at least partially stochastic polynucleotide sequences and translating said population of stochastic polynucleotide sequences to produce said population of peptides, polypeptides or proteins.

9. The method of claim 7 or 8, further comprising amplification of said population of stochastic polynucleotide sequences.

10. The method of claim 6, wherein step (a) further comprises synthesizing a population of stochastic polynucleotide sequences and expressing said population of stochastic polynucleotide sequences to produce said population of peptides, polypeptides or proteins.

11. The method of claim 6, wherein step (a) further comprises synthesizing a population of at least partially stochastic polynucleotide sequences and expressing said population of stochastic polynucleotide sequences to produce said population of peptides, polypeptides or proteins.

12. The method of claim 6, further comprising isolating the polynucleotide sequence encoding said one or more peptides, polypeptides or proteins having said predetermined property.

13. The method of claim 6, wherein said predetermined property comprises binding or chemical catalysis.

14. The method of claim 13, further comprising improving said predetermined property by in vitro or in vivo mutagenesis.

15. The method of claim 13, wherein said binding further comprises modification of a biological or chemical property of a compound bound by said peptide, polypeptide or protein.

16. The method of claim 15, wherein said modification of said biological or chemical property of said compound further comprises stimulating or inhibiting at least one biological function of said compound.

39. The method of claim 6, wherein said population of peptides, polypeptides or proteins comprises greater than about 1×

10⁴ different amino acid sequences.

40. The method of claim 6, wherein said population of peptides, polypeptides or proteins comprises greater than about 1×

10⁵ different amino acid sequences.

41. The method of claim 6, wherein said population of peptides, polypeptides or proteins comprises greater than about 1×

10⁶ different amino acid sequences.

42. The method of claim 6, wherein said population of peptides, polypeptides or proteins comprises greater than about 1×

10⁷ different amino acid sequences.

43. The method of claim 6, wherein said population of peptides, polypeptides or proteins comprises greater than about 1×

10⁸ different amino acid sequences.

17. A method of producing a peptide, polypeptide or protein having a predetermined property, comprising:

(a) producing a population of peptides, polypeptides or proteins encoded by stochastic polynucleotide sequences;

(b) screening said population of peptides, polypeptides or proteins for said predetermined property under conditions which allow detection of one or more peptides, polypeptides or proteins having said predetermined property;

(c) isolating the polynucleotide sequence encoding said one or more peptides, polypeptides or proteins having said predetermined property; and

(d) producing said peptide polypeptide or protein.

18. The method of claim 17, wherein step (a) further comprises synthesizing a population of stochastic polynucleotide sequences and translating said population of stochastic polynucleotide sequences to produce said population of peptides, polypeptides or proteins.

19. The method of claim 17, wherein step (a) further comprises synthesizing a population of at least partially stochastic polynucleotide sequences and translating said population of stochastic polynucleotide sequences to produce said population of peptides, polypeptides or proteins.

20. The method of claim 18 or 19, further comprising amplification of said population of stochastic polynucleotide sequences.

21. The method of claim 17, wherein step (a) further comprises synthesizing a population of stochastic polynucleotide sequences and expressing said population of stochastic polynucleotide sequences to produce said population of peptides, polypeptides or proteins.

22. The method of claim 17, wherein step (a) further comprises synthesizing a population of at least partially stochastic polynucleotide sequences and expressing said population of stochastic polynucleotide sequences to produce said population of peptides, polypeptides or proteins.

23. The method of claim 17, further comprising isolating the polynucleotide sequence encoding said one or more peptides, polypeptides or proteins having said predetermined property.

24. The method of claim 17, wherein said producing in step (d) further comprises chemical synthesis or recombinant expression.

25. The method of claim 17, wherein said predetermined property comprises binding or chemical catalysis.

26. The method of claim 25, further comprising improving said predetermined property by in vitro or in vivo mutagenesis.

27. The method of claim 25, wherein said binding further comprises modification of a biological or chemical property of a compound bound by said peptide, polypeptide or protein.

28. The method of claim 25, wherein said modification of said biological or chemical property of said compound further comprises stimulating or inhibiting at least one biological function of said compound.

44. The method of claim 17, wherein said population of peptides, polypeptides or proteins comprises greater than about 1×

10⁴ different amino acid sequences.

45. The method of claim 17, wherein said population of peptides, polypeptides or proteins comprises greater than about 1×

10⁵ different amino acid sequences.

46. The method of claim 17, wherein said population of peptides, polypeptides or proteins comprises greater than about 1×

10⁶ different amino acid sequences.

47. The method of claim 17, wherein said population of peptides, polypeptides or proteins comprises greater than about 1×

10⁷ different amino acid sequences.

48. The method of claim 17, wherein said population of peptides, polypeptides or proteins comprises greater than about 1×

10⁸ different amino acid sequences.

29. A method of producing a stochastic polynucleotide population, comprising synthesizing stochastic polynucleotide sequences.

30. The method of claim 29, further comprising chemical synthesis.

31. The method of claim 29, further comprising enzymatic synthesis.

32. The method of claim 29, further comprising joining oligonucleotide building blocks.

33. The method of claim 29, further comprising cleaving said population of stochastic polynucleotide sequences.

34. The method of claim 33, further comprising ligating said cleaved population of stochastic polynucleotide sequences to produce a new ensemble of stochastic polynucleotide sequences.

49. The method of claim 29, wherein said stochastic polynucleotide population comprises greater than about 1×

10⁴ different polynucleotide sequences.

50. The method of claim 29, wherein said stochastic polynucleotide population comprises greater than about 1×

10⁵ different polynucleotide sequences.

51. The method of claim 29, wherein said stochastic polynucleotide population comprises greater than about 1×

10⁶ different polynucleotide sequences.

52. The method of claim 29, wherein said stochastic polynucleotide population comprises greater than about 1×

10⁷ different polynucleotide sequences.

53. The method of claim 29, wherein said stochastic polynucleotide population comprises greater than about 1×

10⁸ different polynucleotide sequences.

35. A method of producing a desired compound, comprising combining a population of peptides, polypeptides or proteins encoded by stochastic polynucleotide sequences with two or more reactant precursors under conditions favorable for said precursors to react, and incubating said population of peptides, polypeptides or proteins with said reactant precursors for sufficient time so as to allow the catalysis of said desired compound.

36. A method of identifying a population of peptides, polypeptides or proteins which catalyze a sequence of chemical reactions, comprising:

(a) combining a population of peptides, polypeptides or proteins encoded by stochastic polynucleotide sequences with two or more reactant precursors under conditions favorable for said precursors to react;

(b) incubating said population of peptides, polypeptides or proteins with said reactant precursors for sufficient time to allow the catalysis of said sequence of chemical reactions, and

(c) determining the presence or absence of a compound produced by said sequence of chemical reactions, the presence of said compound indicating that said population of peptides, polypeptides or proteins can catalyze said sequence of chemical reactions.

37. The method of claim 36, further comprising the steps:

(d) dividing the population of peptides, polypeptides or proteins that can catalyze said sequence of chemical reactions into two or more subpopulations;

(e) repeating steps (a) through (c), and

(f) determining the subpopulation which catalyzes the sequence of chemical reactions.

38. The method of claim 37, wherein steps (d) through (f) are repeated one or more times.