Methods and compositions for polypeptide engineering

• US 6,613,514 B2
• Filed: 04/27/2000
• Issued: 09/02/2003
• Est. Priority Date: 02/17/1994
• Status: Expired due to Fees

• Current Assignee: Codexis, Inc.
• Original Assignee: Maxygen Inc.
• Inventors: Patten, Phillip A., Stemmer, Willem P. C.
• Primary Examiner(s): Zitomer, Stephanie W.
• Application Number:
  US09/559,671
• Publication Number:
  US 20020051976A1
• Time in Patent Office: 1,223 Days
• Field of Search: 435/6, 435/69.1, 435/92.1, 435/194, 536/23.1, 536/24.3, 530/350, 530/300
• US Class Current: 435/6.14
• CPC Class Codes: A61K 38/45 Transferases (2)
• CPC Class Codes: A61K 48/00 Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
• CPC Class Codes: C07K 14/43595 from coelenteratae, e.g. medusae
• CPC Class Codes: C07K 14/545 IL-1
• CPC Class Codes: C07K 14/56 IFN-alpha
• 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 15/10 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 15/1027 by DNA shuffling, e.g. RSR, STEP, RPR
• CPC Class Codes: C12N 15/1031 mutagenesis by gene assembly, e.g. assembly by oligonucleotide extension PCR
• 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 15/1058 Directional evolution of libraries, e.g. evolution of libraries is achieved by mutagenesis and screening or selection of mixed population of organisms
• 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 15/67 General methods for enhancing the expression
• CPC Class Codes: C12N 2795/14043 viral genome or elements thereof as genetic vector
• CPC Class Codes: C12N 2795/14143 viral genome or elements thereof as genetic vector
• CPC Class Codes: C12N 7/00 Viruses; Bacteriophages; Compositions thereof; Preparation or purification thereof preparing medicinal viral antigen or antibody compositions, e.g. virus vaccines, A61K39/00
• CPC Class Codes: C12N 9/0069 : acting on single donors with incorporation of molecular oxygen, i.e. oxygenases (1.13)
• CPC Class Codes: C12N 9/1007 : Methyltransferases (general) (2.1.1.)
• CPC Class Codes: C12N 9/16 : acting on ester bonds (3.1)
• CPC Class Codes: C12N 9/86 : acting on amide bonds in cyclic amides, e.g. penicillinase (3.5.2)
• CPC Class Codes: C12Q 1/6811 : Selection methods for production or design of target specific oligonucleotides or binding molecules
• CPC Class Codes: C12Q 1/6853 : using modified primers or templates
• CPC Class Codes: C12Q 1/686 : Polymerase chain reaction [PCR]
• 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
• CPC Class Codes: G01N 33/68 : involving proteins, peptides or amino acids involving lipoproteins G01N33/92
• CPC Class Codes: G01N 33/6845 : Methods of identifying protein-protein interactions in protein mixtures

Abstract

Methods are provided for the evolution of proteins of industrial and pharmaceutical interest, including methods for effecting recombination and selection. Compositions produced by these methods are also disclosed.

Claims

1. A method of screening or selecting a further evolved polypeptide for a desired property, the method comprising:

(a) providing a mixture of nucleic acid subsequences of two or more parental nucleic acid substrate molecules encoding homologous polypeptides, wherein each parental nucleic acid substrate molecule differs from at least one other parental nucleic acid substrate molecule in at least one nucleotide;

(b) ligating two or more of the nucleic acid subsequences together in an ordered reassembly to produce a library of recombinant nucleic acid substrate molecules encoding varients of the homologous polypeptides, which library comprises a plurality of nucleic acid molecules, each member of the plurality comprising at least one subsequence from at least two of the two or more parental nucleic acid substrate molecules, wherein the plurality of nucleic acid molecules are homologous to one another and to the parental substrate nucleic acid molecules;

(c) expressing the library of recombinant nucleic acid substrate molecules to provide evolved polypeptides;

(d) screening or selecting the evolved polypeptides to identify at least one evolved polypeptide with the desired property;

(e) recovering at least one recombinant nucleic acid substrate molecule encoding the at least one evolved polypeptide with the desired property;

(f) recombining the at least one recombinant nucleic acid substrate molecule of (e) with at least one polynucleotide to produce at least one further evolved nucleic acid substrate molecule;

(g) expressing the at least one further evolved nucleic acid substrate molecule to provide at least one further evolved polypeptide; and

,

(h) screening or selecting the at least one further evolved polypeptide for the desired property.

2. The method of claim 1, wherein the mixture of nucleic acid subsequences is provided by primer extension.

3. The method of claim 1, wherein the nucleic acid subsequences are provided in single-stranded form.

4. The method of claim 1, wherein the nucleic acid subsequences of step (a) are random fragments of the two or more parental nucleic acid substrate molecules.

5. The method of claim 1, wherein the nucleic acid subsequences of step (a) are defined segments of the two or more parental nucleic acid substrate molecules.

6. The method of claim 1, wherein the two or more parental nucleic acid substrate molecules comprise DNA substrate molecules.

7. The method of claim 1, wherein the two or more parental nucleic acid substrate molecules comprise single-stranded substrate molecules.

8. The method of claim 1, wherein the two or more parental nucleic acid substrate molecules comprise allelic or species variants of one another.

9. The method of claim 1, wherein at least one of the two or more parental nucleic acid substrate molecules is a mutagenized variant.

10. The method of claim 1, wherein the two or more parental nucleic acid substrate molecules each comprise a gene cluster.

11. The method of claim 1, wherein the two or more parental nucleic acid substrate molecules each comprise at least one recognition site for a restriction endonuclease that generates non-palindromic ends at cleavage sites.

12. The method of claim 1, wherein the two or more parental nucleic acid substrate molecules are members of a gene family.

13. The method of claim 1, wherein the homologous polypeptides encoded by the parental nucleic acid substrate molecules of step (a) are selected from the group consisting of proteins from Table I and alpha interferon.

14. The method of claim 1, wherein at least one member of the library of recombinant nucleic acid substrate molecules of step (b) comprises a full length gene.

15. The method of claim 1, wherein the two or more parental nucleic acid substrate molecules comprise at least about 50% sequence identity.

16. The method of claim 15, wherein the two or more parental nucleic acid substrate molecules comprise at least about 70% sequence identity.

17. The method of claim 16, wherein the two or more parental nucleic acid substrate molecules comprise at least about 80% sequence identity.

18. The method of claim 17, wherein the two or more parental nucleic acid substrate molecules comprise at least about 90% sequence identity.

19. The method of claim 17, wherein in step (a) the nucleic acid subsequences are provided by fragmenting the two or more parental nucleic acid substrate molecules.

20. The method of claim 19, wherein in step (a) the two or more parental nucleic acid substrate molecules are fragmented by nuclease digestion.

21. The method of claim 20, wherein in step (a) the two or more parental nucleic acid substrate molecules are DNA and the nuclease is a DNAse.

22. The method of claim 20, wherein the nuclease comprises a restriction endonuclease.

23. The method of claim 1, wherein the nucleic acid subsequences are from 5 bp to 5 kb in size.

24. The method of claim 23, wherein the nucleic acid subsequences are from 10 bp to 1000 bp in size.

25. The method of claim 24, wherein the nucleic acid subsequences are from 20 bp to 500 bp in size.

26. The method of claim 1, wherein the at least one recombinant nucleic acid substrate molecule recovered in (e) comprises a library of recombinant substrate molecules.

27. The method of claim 1, wherein the at least one recombinant nucleic acid substrate molecule recovered in (e) is subjected to mutagenesis to provide at least one mutagenized nucleic acid substrate molecule.

28. The method of claim 27, wherein the mutagenesis comprises one or more of:

recursive sequence recombination, PCR mutagenesis, oligonucleotide-directed mutagenesis, or site-directed mutagenesis.

29. The method of claim 1, wherein for step (c) the library of recombinant nucleic acid substrate molecules is expressed in a cell population.

80. The method of claim 22, wherein the restriction endonuclease generates non-palindromic ends at cleavage sites.

30. A method of identifying an evolved polypeptide with a desired property, the method comprising:

(a) providing a population of nucleic acid subsequences of two or more parental nucleic acid substrate molecules encoding homologous polypeptides, wherein at least one of the two or more parental nucleic acid substrate molecules is a mutagenized variant;

(b) ligating two or more of the nucleic acid subsequences together in an ordered reassembly to produce a library of recombinant nucleic acid subsequences molecules encoding variants of the homologous polypeptides, which library comprises a plurality of nucleic acid molecules, each member of the plurality comprising at least one subsequence from at least two of the two or more parental nucleic acid substrate molecules, wherein the plurality of nucleic acid molecules are homologous to one another and to the parental substrate nucleic acid molecules;

(c) expressing the library of recombinant nucleic acid substrate molecules to provide evolved polypeptides; and

,

(d) screening or selecting the evolved polypeptides to identify at least one evolved polypeptide with the desired property.

31. The method of claim 30, wherein the nucleic acid subsequences are provided in single-stranded form.

32. The method of claim 30, wherein the nucleic acid subsequences comprise defined segments of the two or more parental nucleic acid substrate molecules of step (a).

33. The method of claim 30, wherein the two or more parental nucleic acid substrate molecules of step (a) comprise DNA substrate molecules.

34. The method of claim 30, wherein the two or more parental nucleic acid substrate molecules of step (a) comprise single-stranded substrate molecules.

35. The method of claim 30, wherein the two or more parental nucleic acid substrate molecules of step (a) comprise allelic or species variants of one another.

36. The method of claim 30, wherein the two or more parental nucleic acid substrate molecules of step (a) each comprise a gene cluster.

37. The method of claim 30, wherein the two or more parental nucleic acid substrate molecules of step (a) are members of a gene family.

38. The method of claim 30, wherein the homologous polypeptides encoded by the parental nucleic acid substrate molecules of step (a) are selected from the group consisting of proteins from Table I and alpha interferon.

39. The method of claim 30, wherein at least one member of the library of recombinant nucleic acid substrate molecules comprises a full length gene.

40. The method of claim 30, wherein for step (c) the library of recombinant nucleic acid substrate molecules is expressed in a cell population.

41. The method of claim 30, wherein in step (a) the nucleic acid subsequences are provided by fragmenting the two or more parental nucleic acid substrate molecules.

42. The method of claim 41, wherein in step (a) the two or more parental nucleic acid substrate molecules are fragmented by nuclease digestion.

43. The method of claim 42, wherein in step (a) the two or more parental nucleic acid substrate molecules are DNA and the nuclease is a DNAse.

44. The method of claim 42, wherein the nuclease comprises a restriction endonuclease.

45. The method of claim 30, wherein the two or more parental nucleic acid substrate molecules comprise at least about 50% sequence identity.

46. The method of claim 45, wherein the two or more parental nucleic acid substrate molecules comprise at least about 70% sequence identity.

47. The method of claim 46, wherein the two or more parental nucleic acid substrate molecules comprise at least about 80% sequence identity.

48. The method of claim 47, wherein the two or more parental nucleic acid substrate molecules comprise at least about 90% sequence identity.

49. The method of claim 30, wherein the nucleic acid subsequences axe from 5 bp to 5 kb in size.

50. The method of claim 49, wherein the nucleic acid subsequences are from 10 bp to 1000 bp in size.

51. The method of claim 50, wherein the nucleic acid subsequences are from 20 bp to 500 bp in size.

52. The method of claim 30, further comprising:

(e) recovering at least one recombinant nucleic acid substrate molecule encoding the at least one evolved polypeptide with the desired property; and

,

(f) recombining the at least one recombinant nucleic acid substrate molecule of (e) with at least one polynucleotide to produce at least one further evolved nucleic acid substrate molecule.

53. The method of claim 52, wherein the at least one recombinant nucleic acid substrate molecule recovered in (e) comprises a library of recombinant substrate molecules.

54. The method of claim 52, wherein the at least one recombinant nucleic acid substrate molecule recovered in (e) is subjected to mutagenesis to provide at least one mutagenized nucleic acid substrate molecule.

55. The method of claim 54, wherein the mutagenesis comprises one or more of:

recursive sequence recombination, PCR mutagenesis, oligonucleotide-directed mutagenesis, or site-directed mutagenesis.

81. The method of claim 44, wherein the restriction endonuclease generates non-palindromic ends at cleavage sites.

56. A method of screening or selecting a further evolved polypeptide for a desired property, the method comprising:

(a) providing a population of double-stranded nucleic acid subsequences of two or more polynucleotides encoding variants of a polypeptide, wherein each double-stranded nucleic subsequence comprises one or more single-stranded overhangs that promote reassembly of the subsequences by ligation;

(b) ligating two or more of the double-stranded nucleic acid subsequences together in an ordered reassembly to produce a library of recombined nucleic acid sequences encoding evolved variants of the polypeptide;

(c) expressing the recombined nucleic acid sequences to produce the evolved variants of the polypeptide;

(d) screening or selecting the evolved variants of the polypeptide produced in step (c) to identify at least one evolved variant of the polypeptide comprising the desired property;

(e) recovering at least one recombined nucleic acid sequence encoding the at least one evolved variant of the polypeptide comprising the desired property;

(f) recombining the at least one recombined nucleic acid sequence of (e) with at least one nucleic acid substrate molecule to produce at least one further evolved nucleic acid sequence;

(g) expressing the at least one further evolved nucleic acid sequence to provide at least one further evolved polypeptide; and

,

(h) screening or selecting the at least one further evolved polypeptide for the desired property.

57. The method of claim 56, wherein the double-stranded nucleic acid subsequences comprise defined segments of the two or more polynucleotides of step (a).

58. The method of claim 56, wherein the two or more polynucleotides of step (a) comprise DNA substrate molecules.

59. The method of claim 56, wherein the two or more polynucleotides of step (a) comprise allelic or species variants of one another.

60. The method of claim 56, wherein the two or more polynucleotides of step (a) each comprise a gene cluster.

61. The method of claim 56, wherein the two or more polynucleotides of step (a) are members of a gene family.

62. The method of claim 56, wherein the variants of the polypeptide encoded by the two or more polynucleotides of step (a) are selected from the group consisting of proteins from Table I and alpha interferon.

63. The method of claim 56, wherein at least one member of the library of recombined nucleic acid sequences comprises a full length gene.

64. The method of claim 56, wherein for step (c) the library of recombined nucleic acid sequences is expressed in a cell population.

65. The method of claim 56, wherein in step (a) the double-stranded nucleic acid subsequences are provided by fragmenting the two or more polynucleotides.

66. The method of claim 65, wherein in step (a) the two or more polynucleotides are fragmented by nuclease digestion.

67. The method of claim 66, wherein in step (a) the two or more polynucleotides are DNA and the nuclease is a DNAse.

68. The method of claim 66, wherein the nuclease comprises a restriction endonuclease.

69. The method of claim 68, wherein the restriction endonuclease generates non-palindromic ends at cleavage sites.

70. The method of claim 56, wherein the two or more polynucleotides comprise at least about 50% sequence identity.

71. The method of claim 70, wherein the two or more polynucleotides comprise at Least about 70% sequence identity.

72. The method of claim 71, wherein the two or more polynucleotides comprise at least about 80% sequence identity.

73. The method of claim 72, wherein the two or more polynucleotides comprise at least about 90% sequence identity.

74. The method of claim 56, wherein the double-stranded nucleic acid subsequences are from 5 bp to 5 kb in size.

75. The method of claim 74, wherein the double-stranded nucleic acid subsequences are from 10 bp to 1000 bp in size.

76. The method of claim 75, wherein the double-stranded nucleic acid subsequences are from 20 bp to 500 bp in size.

77. The method of claim 56, wherein the at least one recombined nucleic acid sequence recovered in (e) comprises a library of recombined nucleic acid sequences.

78. The method of claim 56, wherein the at Least one recombined nucleic acid sequence recovered in (e) is subjected to mutagenesis to provide at least one mutagenized nucleic acid sequence.

79. The method of claim 78, wherein the mutagenesis comprises one or more of:

recursive sequence recombination, PCR mutagenesis, oligonucleotide-directed mutagenesis, or site-directed mutagenesis.