Methods for generating polynucleotides having desired characteristics by iterative selection and recombination

• US 6,506,602 B1
• Filed: 10/25/2000
• Issued: 01/14/2003
• Est. Priority Date: 03/25/1996
• Status: Expired due to Fees

• Current Assignee: Codexis, Inc.
• Original Assignee: Maxygen Inc.
• Inventors: Stemmer, Willem P. C.
• Primary Examiner(s): Whisenant, Ethan C.
• Application Number:
  US09/696,322
• Time in Patent Office: 811 Days
• Field of Search: 435/440, 435/6, 536/23.1, 536/24.3, 935/76, 935/77, 935/78
• US Class Current: 506/1
• CPC Class Codes: C07K 14/43595 from coelenteratae, e.g. medusae
• CPC Class Codes: C07K 14/545 IL-1
• CPC Class Codes: C12N 15/1037 Screening libraries presented on the surface of microorganisms, e.g. phage display, E. coli display
• CPC Class Codes: C12N 15/52 Genes encoding for enzymes or proenzymes
• 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 9/2471 Beta-galactosidase (3.2.1.23), i.e. exo-(1-->4)-beta-D-galactanase
• CPC Class Codes: C12N 9/86 acting on amide bonds in cyclic amides, e.g. penicillinase (3.5.2)
• CPC Class Codes: C12Y 302/01023 Beta-galactosidase (3.2.1.23), i.e. exo-(1-->4)-beta-D-galactanase
• CPC Class Codes: C40B 40/02 Libraries contained in or displayed by microorganisms, e.g. bacteria or animal cells; Libraries contained in or displayed by vectors, e.g. plasmids; Libraries containing only microorganisms or vectors

Abstract

A method for DNA reassembly after random fragmentation, and its application to mutagenesis of nucleic acid sequences by in vitro or in vivo recombination is described. In particular, a method for the production of nucleic acid fragments or polynucleotides encoding mutant proteins is described. The present invention also relates to a method of repeated cycles of mutagenesis, shuffling and selection which allow for the directed molecular evolution in vitro or in vivo of proteins.

Claims

1. A method of generating a combinatorial library of recombinant nucleic acids, the method comprising:

(a) providing a plurality of non-identical deoxyribonucleic acids comprising intron and exon sequences, which intron and exon sequences comprise a plurality of different splicing frames; and

,

(b) recombining the plurality of non-identical deoxyribonucleic acids to produce predominantly in frame combinations of the exon sequences, thereby providing the combinatorial library.

2. The method of claim 1, wherein the plurality of non-identical deoxyribonucleic acids comprise a polynucleotide encoding a protein.

3. The method of claim 2, wherein the protein comprises an enzyme.

4. The method of claim 2, wherein the protein comprises a therapeutic protein.

5. The method of claim 1, the plurality of non-identical deoxyribonucleic acids comprising at least one naturally occurring gene.

6. The method of claim 1, comprising providing a plurality of nucleic acid comprising at least one naturally occurring intron.

7. The method of claim 1, comprising providing a plurality of nucleic acid comprising at least one synthetic intron.

8. The method of claim 1, wherein the exons lack sequence identity.

9. The method of claim 1, wherein the exons comprise random sequence exons.

10. The method of claim 1, comprising recombining sequences of at least two distinct members of the plurality of non-identical deoxyribonucleic acids.

14. The method of claim 1, further comprising screening or selecting at least one member of the combinatorial library for a desired property.

15. The method of claim 1, further comprising recombining at least one member of the combinatorial library with one or more additional nucleic acids.

16. The method of claim 15, wherein the recombining with the one or more additional nucleic acids is performed in vitro.

17. The method of claim 15, wherein the recombining with the one or more additional nucleic acids is performed in vivo.

11. The method of 10, comprising recombining intron sequences.

12. The method of 10, wherein the recombining comprises homologous recombination or site specific recombination between the distinct members.

13. A method of generating a combinatorial library of recombinant nucleic acids, the method comprising:

(a) providing a plurality of nucleic acids comprising intron and exon sequences, which intron and exon sequences comprise a plurality of splicing frames; and

,

(b) recombining the plurality of nucleic acids to produce predominantly in frame combinations of the exon sequences, thereby providing the combinatorial library, wherein the predominantly in frame combinations of exon sequences are produced by concatamerizing a plurality of exons during a polymerase chain reaction (PCR) using at least one primer that spans intron segments.

18. A method of generating a combinatorial library of recombinant nucleic acids, the method comprising:

(a) providing a plurality of non-identical deoxyribonucleic acids comprising intron and exon sequences, which intron and exon sequences comprise a plurality of different splicing frames and the intron and exon sequences are selected to predominantly comprise a single splicing frame; and

,

(b) recombining the plurality of non-identical deoxyribonucleic acids to produce a plurality of in frame combinations of the exon sequences, thereby providing the combinatorial library.

19. The method of claim 18, wherein the plurality of non-identical deoxyribonucleic acids comprise a polynucleotide encoding a protein.

20. The method of claim 19, wherein the protein comprises an enzyme.

21. The method of claim 19, wherein the protein comprises a therapeutic protein.

22. The method of claim 18, the plurality of non-identical deoxyribonucleic acids comprising at least one naturally occurring gene.

23. The method of claim 18, comprising providing a plurality of nucleic acid comprising at least one naturally occurring intron.

24. The method of claim 18, comprising providing a plurality of nucleic acid comprising at least one synthetic intron.

25. The method of claim 18, wherein at least one of the plurality of non-identical deoxyribonucleic acids comprises a splicing module comprising multiple exons, which exons have the same splicing frame at each end.

26. The method of claim 18, wherein the exons lack sequence identity.

27. The method of claim 18, wherein the exons comprise random sequence exons.

28. The method of claim 18, comprising recombining sequences of at least two distinct members of the plurality of non-identical deoxyribonucleic acids.

29. The method of claim 28, comprising recombining intron sequences.

30. The method of claim 28, wherein the recombining comprises homologous recombination or site specific recombination between the distinct members.

31. The method of claim 18, further comprising screening or selecting at least one member of the combinatorial library for a desired property.

32. The method of claim 18, further comprising recombining at least one member of the combinatorial library with one or more additional nucleic acids.

33. The method of claim 32, wherein the recombining with the one or more additional nucleic acids is performed in vitro.

34. The method of claim 32, wherein the recombining with the one or more additional nucleic acids is performed in vivo.

35. A method of generating a combinatorial library of recombinant nucleic acids, the method comprising:

(a) providing a plurality of non-identical deoxyribonucleic acids comprising exon sequences, wherein the exon sequences comprise a plurality of different splicing frames; and

(b) recombining the plurality of non-identical deoxyribonucleic acids to produce predominantly in frame combinations of the exon sequences, thereby providing the combinatorial library.

36. The method of claim 35, wherein at least one of the plurality of non-identical deoxyribonucleic acids comprises a splicing module comprising multiple exons, which exons have the same splicing frame at each end.

37. The method of claim 35, wherein the plurality of non-identical deoxyribonucleic acids comprise a polynucleotide encoding a protein.

38. The method of claim 37, wherein the protein comprises an enzyme.

39. The method of claim 37, wherein the protein comprises a therapeutic protein.

40. The method of claim 35, the plurality of non-identical deoxyribonucleic acids comprising at least one naturally occurring gene.

41. The method of claim 35, comprising providing a plurality of nucleic acid further comprising at least one naturally occurring intron.

42. The method of claim 35, comprising providing a plurality of nucleic acid further comprising at least one synthetic intron.

43. The method of claim 35, wherein the exons lack sequence identity.

44. The method of claim 35, comprising recombining sequences of at least two distinct members of the plurality of non-identical deoxyribonucleic acids.

45. The method of claim 44, wherein the recombining comprises homologous recombination or site specific recombination between the distinct members.

47. The method of claim 35, further comprising screening or selecting at least one member of the combinatorial library for a desired property.

48. The method of claim 35, further comprising recombining at least one member of the combinatorial library with one or more additional nucleic acids.

49. The method of claim 48, wherein the recombining with the one or more additional nucleic acids is performed in vitro.

50. The method of claim 48, wherein the recombining with the one or more additional nucleic acids is performed in vivo.

46. A method of generating a combinatorial library of recombinant nucleic acids, the method comprising:

(a) providing a plurality of nucleic acids comprising exon sequences, wherein the exon sequences comprise one or more splicing frames; and

(b) recombining the plurality of nucleic acids to produce predominantly in frame combinations of the exon sequences, thereby providing the combinatorial library, wherein the predominantly in frame combinations of exon sequences are produced by concatamerizing a plurality of exons during a polymerase chain reaction (PCR).

51. A method of generating a combinatorial library of recombinant nucleic acids, the method comprising:

(a) providing a plurality of non-identical deoxyribonucleic acids comprising splicing modules, wherein the splicing modules comprise a plurality of different splicing frames; and

(b) recombining the plurality of non-identical deoxyribonucleic acids to produce predominantly in frame combinations of the splicing modules, thereby providing the combinatorial library.

52. The method of claim 51, comprising providing a plurality of non-identical deoxyribonucleic acids comprising splicing modules, wherein the splicing modules are selected to predominantly comprise a single splicing frame.

53. The method of claim 51, wherein the splicing modules begin and end in the same splicing frame.

54. The method of claim 51, wherein the splicing modules comprise one or more exons.

55. The method of claim 54, wherein at least one of the splicing modules comprises multiple exons, which exons have the same splicing frame at each end.

56. The method of claim 51, wherein the splicing module comprises one or more protein domains.

57. The method of claim 56, wherein the splicing module comprises at least one immunoglobulin variable domain.

58. The method of claim 51, wherein the plurality of non-identical deoxyribonucleic acids comprise a polynucleotide encoding a protein.

59. The method of claim 58, wherein the protein comprises an enzyme.

60. The method of claim 58, wherein the protein comprises a therapeutic protein.

61. The method of claim 51, the plurality of non-identical deoxyribonucleic acids comprising at least one naturally occurring gene.

62. The method of claim 51, comprising providing a plurality of nucleic acid further comprising at least one naturally occurring intron.

63. The method of claim 51, comprising providing a plurality of nucleic acid further comprising at least one synthetic intron.

64. The method of claim 51, wherein the exons lack sequence identity.

65. The method of claim 51, omprising recombining sequences of at least two distinct members of the plurality of non-identical deoxyribonucleic acids.

66. The method of claim 65, wherein the recombining comprises homologous recombination or site specific recombination between the distinct members.

68. The method of claim 51, further comprising screening or selecting at least one member of the combinatorial library for a desired property.

69. The method of claim 51, further comprising recombining at least one member of the combinatorial library with one or more additional nucleic acids.

70. The method of claim 69, wherein the recombining with the one or more additional nucleic acids is performed in vitro.

71. The method of claim 69, wherein the recombining with the one or more additional nucleic acids is performed in vivo.

67. A method of generating a combinatorial library of recombinant nucleic acids, the method comprising:

(a) providing a plurality of nucleic acids comprising splicing modules, wherein the splicing modules comprise one or more splicing frame; and

(b) recombining the plurality of nucleic acid to produce predominantly in frame combinations of the splicing module sequences, thereby providing the combinatorial library, wherein the predominantly in frame combinations of exon sequences are produced by concatamerizing a plurality of exons during a polymerase chain reaction (PCR).