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

• US 6,287,861 B1
• Filed: 08/12/1998
• Issued: 09/11/2001
• Est. Priority Date: 02/17/1994
• Status: Expired due to Term

• Current Assignee: Codexis, Inc.
• Original Assignee: Maxygen Inc.
• Inventors: Stemmer, Willem P. C., Crameri, Andreas
• Primary Examiner(s): WHISENANT, ETHAN C
• Application Number:
  US09/133,508
• Time in Patent Office: 1,126 Days
• Field of Search: 435/6, 435/172.3, 435/400, 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: C07K 16/00 Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies antibodies with enzymatic activity, e.g. abzymes C12N9/0002
• CPC Class Codes: C07K 2317/565 Complementarity determining region [CDR]
• CPC Class Codes: C07K 2317/622 Single chain antibody (scFv)
• CPC Class Codes: C12N 15/1027 by DNA shuffling, e.g. RSR, STEP, RPR
• CPC Class Codes: C12N 15/1034 Isolating an individual clone by screening libraries
• 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 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: C12Q 1/68 involving nucleic acids
• CPC Class Codes: C12Q 1/6811 Selection methods for production or design of target specific oligonucleotides or binding molecules
• CPC Class Codes: C12Y 302/01023 Beta-galactosidase (3.2.1.23), i.e. exo-(1-->4)-beta-D-galactanase
• CPC Class Codes: C12Y 305/02006 Beta-lactamase (3.5.2.6)
• 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
• CPC Class Codes: C40B 40/08 Libraries containing RNA or DNA which encodes proteins, e.g. gene libraries
• CPC Class Codes: C40B 50/06 Biochemical methods, e.g. using enzymes or whole viable microorganisms

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 for evolving a polynucleotide for acquisition of a desired functional property, comprising:

(1) providing a library of variant forms of a polynucleotide, some of which are components of a vector, and some of which are not components of a vector,

(2) introducing the library of variant forms into a population of cells,

(3) propagating the cells under conditions whereby recombination occurs between the variant forms that are components of a vector and the variant forms that are not components of a vector to generate recombinant forms of the polynucleotide;

(4) selecting or screening the recombinant forms of the polynucleotide to identify at least one recombinant form of the polynucleotide having evolved toward a desired property.

2. The method of claim 1, wherein the variant forms that are components of a vector are components of a phage vector.

3. The method of claim 1, wherein the variant forms that are components of a vector are components of a plasmid vector.

4. The method of claim 1, wherein the library of variant forms are introduced by a process selected from the group consisting of electroporation, lipofection, biolisitics, and conjugation.

5. The method of claim 1, wherein the variant forms that are components of a vector are introduced into cells before the variant forms that are not components of a vector.

6. The method of claim 1, further comprising isolating the recombinant forms and repeating steps (1)-(4) wherein the library of variant forms comprises the recombinant forms.

7. The method of claim 1, wherein the variants of the polynucleotide are naturally occurring variants.

8. The method of claim 7, wherein the naturally occurring variant of the polynucleotide encode naturally occurring polypeptides.

9. The method of claim 7, wherein the naturally occurring variants of a polynucleotide comprise human polynucleotides.

10. The method of claim 7, wherein the naturally occurring variants of a polynucleotide comprise bacterial polynucleotides.

11. The method of claim 7, wherein the naturally occurring variants of a polynucleotide comprise a plant polynucleotides.

12. The method of claim 7, wherein the naturally occurring variants of a polynucleotide comprise a fungal polynucleotides.

13. The method of claim 7, wherein the naturally occurring variants of a polynucleotide comprise animal polynucleotides.

14. The method of claim 7, wherein the naturally occurring variants of a polynucleotide comprise viral polynucleotides.

15. The method of claim 7, wherein the naturally occurring variants of a polynucleotide comprise nonallelic gene sequences from the same species having sufficient sequence similarity for annealing of segments of different variants.

16. The method of claim 7, wherein the naturally occurring variants of a polynucleotides comprise allelic variants of a gene.

17. The method of claim 7, wherein the naturally occurring variants of a polynucleotide comprises species variants of a gene.

18. The method of claim 1, wherein the desired property is capacity to bind a receptor.

19. The method of claim 1, wherein the desired property is drug resistance.

20. The method of claim 1, wherein the desired property is a therapeutic activity.

21. The method of claim 1, wherein the recombinant polynucleotide is screened for suitable as an agent for gene therapy.

22. The method of claim 1, wherein the recombinant polynucleotide is screened for suitability as an agent for anti-neoplastic therapy.

23. The method of claim 1, wherein the recombinant polynucleotide is screened for suitability as an agent for DNA-based vaccination.

24. The method of claim 1, further comprising shuffling a recombinant polynucleotide with the desired property with a naturally occurring polynucleotide to eliminate variations between the recombinant polynucleotide and the naturally occurring polynucleotide that do not contribute to the desired property of the recombinant polynucleotide.

25. The method of claim 1, further comprising fragmenting the variants that are not components of a vector before introducing the variants into the cells.

26. The method of claim 25, wherein the variants are randomly fragmented.

27. The method of claim 1, wherein the library of variant forms is provided by conducting a polynucleotide amplification process on overlapping segments of a population of polynucleotides under conditions whereby one segment serves as a template for extension of another segment to generate a population of variant forms.

28. A method for evolving a polynucleotide for acquisition of a desired functional property, comprising:

(1) providing a mixed population of variant forms of a polynucleotide, each form incorporated in a separate copy of the same vector;

(2) introducing the mixed population of variant forms into a population of cells, whereby some cells receive from the mixed population at least two different variant forms of the polynucleotide incorporated on separate copies of the vector, which recombine with each other, thereby forming a library of recombinant forms of the polynucleotide incorporated in separate copies of the vector;

(3) selecting or screening the recombinant forms of the polynucleotides to identify at least one recombinant form of the polynucleotide having evolved toward the desired functional property.

29. The method of claim 28, wherein the vector is a phage vector.

30. The method of claim 28, wherein the vector is a plasmid vector.

31. The method of claim 28, wherein the library of variant forms are introduced by a process selected from the group consisting of electroporation, lipofection, biolistics, and conjugation.

32. The method of claim 28, further comprising isolating recombinant forms having evolved toward the desired functional property and repeating steps (1)-(3) wherein the variant forms comprise the recombinant forms.

33. The method of claim 28, wherein the variants of the polynucleotide are naturally occurring variants.

34. The method of claim 33, wherein the naturally occurring variants of the polynucleotide encode naturally occurring polypeptides.

35. The method of claim 33, wherein the naturally occurring variants of a polynucleotide comprise human polynucleotides.

36. The method of claim 33, wherein the naturally occurring variants of a polynucleotide comprise bacterial polynucleotides.

37. The method of claim 33, wherein the naturally occurring variants of a polynucleotide comprise a plant polynucleotides.

38. The method of claim 33, wherein the naturally occurring variants of a polynucleotide comprise a fungal polynucleotides.

39. The method of claim 33, wherein the naturally occurring variants of a polynucleotide comprise animal polynucleotides.

40. The method of claim 33, wherein the naturally occurring variants of a polynucleotide comprise viral polynucleotides.

41. The method of claim 33, wherein the naturally occurring variants of a polynucleotide comprise nonallelic gene sequences from the same species having sufficient sequence similarity for annealing of segments of different variants.

42. The method of claim 33, wherein the naturally occurring variants of a polynucleotide comprise allelic variants of a gene.

43. The method of claim 33, wherein the naturally occurring variants of a polynucleotide comprises species variants of a gene.

44. The method of claim 28, wherein the desired functional property is capacity to bind a receptor.

45. The method of claim 28, wherein the desired functional property is drug resistance.

46. The method of claim 28, wherein the desired functional property is a therapeutic activity.

47. The method of claim 28, wherein the recombinant polynucleotide is screened for suitability as an agent for gene therapy.

48. The method of claim 28, wherein the recombinant polynucleotide is screened for suitability as an agent for anti-neoplastic therapy.

49. The method of claim 28, wherein the recombinant polynucleotide is screened for suitability as an agent for DNA-based vaccination.

50. The method of claim 28, further comprising shuffling a recombinant polynucleotide with the desired functional property with a naturally occurring polynucleotide to eliminate variations between the recombinant polynucleotide and the naturally occurring polynucleotide that do not contribute to the desired functional property of the recombinant polynucleotide.

51. A method for evolving a polynucleotide for acquisition of a desired functional property, comprising:

(1) providing variant forms of a polynucleotide, each form incorporated in a vector;

(2) introducing the variant forms into a population of cells, whereby some cells receive at least two different variant forms of the polynucleotide, whereby the different variant forms of the polynucleotides recombine to form a library of recombinant forms of the polynucleotide incorporated in the vector;

(3) selecting or screening for recombinant forms of the polynucleotides having evolved toward the desired functional property;

(4) repeating (2) and (3) with further variant forms of the polynucleotide comprising a subset of the recombinant forms having evolved toward the desired functional property, until a recombinant form of a polynucleotide has acquired the desired functional property.

52. The method of claim 51, wherein the vector is a phage vector.

53. The method of claim 51, wherein the vector is a plasmid vector.

54. The method of claim 51, wherein the variant forms of a polynucleotide are introduced by a process selected from the group consisting of electroporation, lipofection, biolistics, and conjugation.

55. The method of claim 51, wherein the variant forms of the polynucleotide are naturally occurring variants.

56. The method of claim 55, wherein the naturally occurring variants of the polynucleotide encode naturally occurring polypeptides.

57. The method of claim 56, wherein the naturally occurring variants of a polynucleotide comprise human polynucleotides.

58. The method of claim 56, wherein the naturally occurring variants of a polynucleotide comprise bacterial polynucleotides.

59. The method of claim 56, wherein the naturally occurring variants of a polynucleotide comprise plant polynucleotides.

60. The method of claim 56, wherein the naturally occurring variants of a polynucleotide comprise fungal polynucleotides.

61. The method of claim 56, wherein the naturally occurring variants of a polynucleotide comprise animal polynucleotides.

62. The method of claim 56, wherein the naturally occurring variants of a polynucleotide comprise viral polynucleotides.

63. The method of claim 56, wherein the naturally occurring variants of a polynucleotide comprise nonallelic gene sequences from the same species having sufficient sequence similarity for annealing of segments of different variants.

64. The method of claim 56, wherein the naturally occurring variants of a polynucleotides comprise allelic variants of a gene.

65. The method of claim 56, wherein the naturally occurring variants of a polynucleotide comprises species variants of a gene.

66. The method of claim 51, wherein the desired functional property is capacity to bind a receptor.

67. The method of claim 51, wherein the desired functional property is drug resistance.

68. The method of claim 51, wherein the desired functional property is a therapeutic activity.

69. The method of claim 51, wherein the recombinant polynucleotide is screened for suitability as an agent for gene therapy.

70. The method of claim 51, wherein the recombinant polynucleotide is screened for suitability as an agent for anti-neoplastic therapy.

71. The method of claim 51, wherein the recombinant polynucleotide is screened for suitability as an agent for DNA-based vaccination.

72. The method of claim 51, further comprising shuffling a recombinant polynucleotide with the desired functional property with a naturally occurring polynucleotide to eliminate variations between the recombinant polynucleotide and the naturally occurring polynucleotide that do not contribute to the desired functional property of the recombinant polynucleotide.

73. A method of evolving a polynucleotide sequence toward a desired functional property, comprising:

(1) recombining variant forms of a polynucleotide sequence in a host cell, at least some of which forms are provided in cell-free form before introduction into the host cell;

(2) selecting or screening for recombinant forms of the polynucleotide sequence that have evolved toward the desired functional property;

(3) recombining selected or screened recombinant forms having evolved toward the desired functional property with each other to generate further recombinant forms;

(4) selecting or screening for further recombinant forms of the polynucleotide sequence that have evolved toward the desired functional property.

74. The method of claim 73, wherein some of the variant forms are stably integrated in the host cell.

75. The method of claim 73, wherein the variant forms in cell-free form are provided as components of a phage vector.

76. The method of claim 73, wherein the variant forms in cell-free form are provided as components of a plasmid vector.

77. The method of claim 73, wherein the variant forms in cell-free form are introduced into the host cell by a process selected from the group consisting of electroporation, lipofection, biolistics, and conjugation.

78. The method of claim 77, wherein the variant forms are naturally occurring variants.

79. The method of claim 78, wherein the naturally occurring variant of the polynucleotide encode naturally occurring polypeptides.

80. The method of claim 78, wherein the naturally occurring variants of a polynucleotide comprise human polynucleotides.

81. The method of claim 78, wherein the naturally occurring variants of a polynucleotide comprise bacterial polynucleotides.

82. The method of claim 78, wherein the naturally occurring variants of a polynucleotide comprise a plant polynucleotides.

83. The method of claim 78, wherein the naturally occurring variants of a polynucleotide comprise fungal polynucleotides.

84. The method of claim 78, wherein the naturally occurring variants of a polynucleotide comprise animal polynucleotides.

85. The method of claim 78, wherein the naturally occurring variants of a polynucleotide comprise viral polynucleotides.

86. The method of claim 78, wherein the naturally occurring variants of a polynucleotide comprise nonallelic gene sequences from the same species having sufficient sequence similarity for annealing of segments of different variants.

87. The method of claim 78, wherein the naturally occurring variants of a polynucleotides comprise allelic variants of a gene.

88. The method of claim 78, wherein the naturally occurring variants of a polynucleotide comprises species variants of a gene.

89. The method of claim 73, wherein the desired functional property is capacity to bind a receptor.

90. The method of claim 73, wherein the desired functional property is drug resistance.

91. The method of claim 73, wherein the desired functional property is a therapeutic activity.

92. The method of claim 73, wherein desired functional property is suitability as an agent for gene therapy.

93. The method of claim 73, wherein the desired functional property is suitability as an agent for anti-neoplastic therapy.

94. The method of claim 73, wherein the desired functional property is suitability as an agent for DNA-based vaccination.