Method of identifying a stochastically-generated peptide, polypeptide, or protein having ligand binding property and compositions thereof

• US 5,723,323 A
• Filed: 12/02/1994
• Issued: 03/03/1998
• 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:
  US08/349,510
• Time in Patent Office: 1,187 Days
• Field of Search: 435/7.1, 435/69.1, 435/91.1, 435/172.3, 536/23.1, 530/300, 530/350
• US Class Current: 435/6.11
• 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 method of identifying a peptide, polypeptide or protein having a binding property to a ligand, comprising:

(a) providing a ligand for detecting said binding property;

(b) synthesizing a diverse population of stochastically generated polynucleotide sequences;

(c) inserting said diverse population of stochastically generated polynucleotide sequences into a population of expression vectors to form a diverse population of expression vectors containing stochastically generated polynucleotide sequences;

(d) expressing in host cells said diverse population of expression vectors containing stochastically generated polynucleotide sequences to produce a diverse population of peptides, polypeptides or proteins; and

(e) screening said diverse population of peptides, polypeptides or proteins with said ligand under conditions which allow binding and detection of one or more peptides, polypeptides or proteins having said predetermined property.

2. The method of claim 1, wherein said stochastically generated polynucleotide sequences further comprises all twenty amino acid residues encoded at each codon position.

3. The method of claim 1, wherein said diverse population of stochastically generated polynucleotide sequences, encode at least 10,000 different peptides, polypeptides or proteins.

4. The method of claim 1, wherein said inserting further comprises hybridization of complementary ends.

5. The method of claim 1, wherein said inserting further comprises ligation.

6. The method of claim 1, wherein said diverse population of stochastically generated polynucleotide sequences are produced by stochastic copolymerization of double stranded oligonucleotides.

7. The method of claim 1, wherein said diverse population of stochastically generated polynucleotide sequences are produced by copolymerization of the four kinds of nucleotide triphosphates consisting of Adenine, Cytosine, Guanine and Thymine.

8. The method of claim 1, wherein said diverse population of stochastically generated polynucleotide sequences are produced by chemical synthesis.

9. The method of claim 1, wherein the expression vector is a plasmid.

10. The method of claim 9, wherein the plasmid is pUC8.

11. The method of claim 1, wherein the expression vector is vital DNA.

12. The method of claim 1, wherein the expression vector is a hybrid of plasmid and vital DNA.

13. The method of claim 1, wherein the expression vector is a phage.

14. The method of claim 1, wherein step (c) further comprises digesting the diverse population of expression vectors with a restriction enzyme having a recognition sequence absent in the expression vector and reinserting the digested products into said digested population of vectors to form a different population having a greater number of new stochastic polynucleotide sequences.

15. An isolated, diverse population of peptides, polypeptides, or proteins comprising greater than about 1×

10⁵ different stochastic amino acid sequences encoded by stochastic polynucleotide sequences.

46. The isolated diverse population of peptides, polypeptides, or proteins of claim 15, further comprising greater than about 1×

10⁶ different stochastic amino acid sequences.

47. The isolated diverse population of peptides, polypeptides, or proteins of claim 15, further comprising greater than about 1×

10⁷ different stochastic amino acid sequences.

48. The isolated, diverse population of peptides, polypeptides, or proteins of claim 15, further comprising greater than about 1×

10⁸ different stochastic amino acid sequences.

16. A method of isolating a polynucleotide sequence encoding a peptide, polypeptide or protein having a predetermined binding property to a ligand, comprising:

(a) providing a ligand for detecting said binding property;

(b) synthesizing a diverse population of stochastically generated polynucleotide sequences;

(c) inserting said diverse population of stochastically generated polynucleotide sequences into a population of expression vectors to form a diverse population of expression vectors containing stochastically generated polynucleotide sequences;

(d) expressing in host cells said diverse population of expression vectors containing stochastically generated polynucleotide sequences to produce a diverse population of peptides, polypeptides or proteins;

(e) screening said diverse population of peptides, polypeptides or proteins with said ligand under conditions which allow binding and detection of one or more peptides, polypeptides or proteins to said ligand; and

(f) isolating the stochastically generated polynucleotide sequence or sequences which encoding said peptides, polypeptides or proteins having said predetermined binding property to said ligand.

17. The method of claim 16, wherein said stochastically generated polynucleotide sequences further comprises all twenty amino acid residues encoded at each codon position.

18. The method of claim 16, wherein said diverse population of stochastically generated polynucleotide sequences, encode at least 10,000 different peptides, polypeptides or proteins.

19. The method of claim 16, wherein said inserting further comprises hybridization of complementary ends.

20. The method of claim 16, wherein said inserting further comprises ligation.

21. The method of claim 16, wherein said diverse population of stochastically generated polynucleotide sequences are produced by stochastic copolymerization of double stranded oligonucleotides.

22. The method of claim 16, wherein said diverse population of stochastically generated polynucleotide sequences are produced by copolymerization of the four kinds of nucleotide triphosphates consisting of Adenine, Cytosine, Guanine and Thymine.

23. The method of claim 16, wherein step (c) further comprises digesting the diverse population of expression vectors with a restriction enzyme having a recognition sequence absent in the expression vector and reinserting the digested products into said digested population of vectors to form a different population having a greater number of new stochastic polynucleotide sequences.

24. An isolated, diverse population of polynucleotide sequences which encode a diverse population of ligand binding peptides, polypeptides, or proteins comprising greater than about 1×

10⁵ different stochastic amino acid sequences encoded by the stochastic polynucleotide sequences.

25. A method of isolating a peptide, polypeptide or protein having a binding property, comprising:

(a), providing a ligand for detecting said binding property;

(b) synthesizing a diverse population of stochastically generated polynucleotide sequences;

(c) inserting said diverse population of stochastically generated polynucleotide sequences into a population of expression vectors to form a diverse population of expression vectors containing stochastically generated polynucleotide sequences;

(d) expressing in host cells said diverse population of expression vectors containing stochastically generated polynucleotide sequences to produce a diverse population of peptides, polypeptides or proteins;

(e) screening said diverse population of peptides, polypeptides or proteins with said ligand under conditions which allow binding and detection of one or more peptides, polypeptides or proteins having said predetermined property;

(f) isolating the stochastically generated polynucleotide sequence or sequences encoding said peptides, polypeptides or proteins having said binding property to said ligand; and

(g) using genetic information from said isolated stochastically generated polynucleotide sequence to produce said peptide, polypeptide or protein having said binding property.

26. The method of claim 25, wherein said stochastically generated polynucleotide sequences further comprises all twenty amino acid residues encoded at each codon position.

27. The method of claim 25, which said diverse population of stochastically generated polynucleotide sequences, encode at least 10,000 different peptides, polypeptides or proteins.

28. The method of claim 25, wherein said inserting further comprises hybridization of complementary ends.

29. The method of claim 25, wherein said inserting further comprises ligation.

30. The method of claim 25, wherein said diverse population of stochastically generated polynucleotide sequences are produced by stochastic copolymerization of double stranded oligonucleotides.

31. The method of claim 25, wherein said diverse population of stochastically generated polynucleotide sequences are produced by copolymerization of the four kinds of nucleotide triphosphates consisting of Adenine, Cytosine, Guanine and Thymine.

32. The method of claim 25, wherein said diverse population of stochastically generated polynucleotide sequences are produced by chemical synthesis.

33. The method of claim 25, wherein step (c) further comprises digesting the diverse population of expression vectors with a restriction enzyme having a recognition sequence absent in the expression vector and reinserting the digested products into said digested population of vectors to form a new different population having a greater number of new stochastic polynucleotide sequences.

34. A method of producing a diverse population of stochastically generated polynucleotide sequences encoding a diverse population of ligand binding peptides, polypeptides or proteins, comprising:

(a) synthesizing a diverse population of stochastically generated polynucleotide sequences selected from a method consisting of stochastic copolymerization of double stranded oligonucleotides, copolymerization of the four kinds of nucleotide triphoshpates consisting of Adenine, Cytosine, Guanine and Thymine, and chemical synthesis; and

(b) inserting said diverse population of stochastically generated polynucleotide sequences into a population of vectors to form a diverse population of vectors containing stochastically generated polynucleotide sequences.

35. The method of claim 34, wherein said stochastically generated polynucleotide sequences further comprises all twenty amino acid residues encoded at each codon position.

36. The method of claim 34, which said diverse population of stochastically generated polynucleotide sequences, encode at least 10,000 different peptides, polypeptides or proteins.

37. The method of claim 34, wherein said inserting further comprises hybridization of complementary ends.

38. The method of claim 34, wherein said inserting further comprises ligation.

39. The method of claim 34, further comprising introducing said diverse population of vectors containing stochastically generated polynucleotide sequences into host cells.

40. The method of claim 34, wherein step (b) further comprises digesting the diverse population of expression vectors with a restriction enzyme having a recognition sequence absent in the expression vector and reinserting the digested products into said digested population of vectors to form a new different population having a greater number of new stochastic polynucleotide sequences.

41. An isolated, diverse population of vectors comprising stochastically generated polynucleotide sequences encoding a diverse population of ligand binding peptides, polypeptides, or proteins comprising greater than about 1×

10⁵ different stochastic amino acid sequences.

42. An isolated, diverse population of peptides or polypeptides comprising stochastic amino acid sequences encoded by stochastic polynucleotide sequences of about 300 nucleotides or less in length.

43. An isolated, diverse population of polynucleotides sequences encoding a diverse population of ligand binding peptides or polypeptides comprising stochastic amino acid sequences encoded by stochastic polynucleotide sequences of about 300 nucleotides or less in length.

44. An isolated, diverse population of vectors encoding a diverse population of ligand binding peptides or polypeptides comprising stochastic amino acid sequences encoded by stochastically generated polynucleotide sequences of about 300 nucleotides or less in length.

45. An isolated, diverse population of peptides, polypeptides, or proteins comprising stochastic amino acid sequences produced by a method comprising synthesizing a diverse population of stochastically generated polynucleotide sequences selected from a method consisting of stochastic copolymerization of double stranded oligonucleotides, copolymerization of the four kinds of nucleotide triphosphates consisting of Adenine, Cytosine, Guanine and Thymine, and chemical synthesis;

and expressing said stochastically generated polynucleotide sequences.