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Last Updated: March 28, 2024

Claims for Patent: 6,335,160


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Summary for Patent: 6,335,160
Title: Methods and compositions for polypeptide engineering
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.
Inventor(s): Patten; Phillip A. (Mountain View, CA), Stemmer; Willem P. C. (Los Gatos, CA)
Assignee: Maxygen, Inc. (Redwood City, CA)
Application Number:08/769,062
Patent Claims:1. A method for producing a recombinant DNA encoding a protein, the method comprising:

(a) digesting at least a first and second DNA substrate molecule, wherein the at least first and second substrate molecules are homologous and differ from each other in at least one nucleotide, with a restriction endonuclease, wherein the at least first and second DNA substrate molecules each encode a protein, or are homologous to a protein-encoding DNA substrate molecule;

(b) ligating the resulting mixture of DNA fragments to generate a library of recombinant DNA molecules, which library comprises a plurality of DNA molecules, each comprising a subsequence from the first nucleic acid and a subsequence from the second nucleic acid, wherein the plurality of DNA molecules are homologous;

(c) screening or selecting the resulting products of (b) for a desired property;

(d) recovering a recombinant DNA molecule encoding an evolved protein; and,

(e) repeating steps a-d using the recombinant DNA molecule of step (d) as the first or second DNA substrate molecule of step (a), whereby a recombinant DNA encoding a protein is produced.

2. The method of claim 1, wherein the restriction endonuclease generates non-palindromic ends at cleavage sites.

3. The method of claim 1, wherein the substrate molecules have been engineered to contain at least one recognition site for a restriction endonuclease having non-palindromic ends at cleavage sites.

4. The method of claim 1, wherein steps (a)-(d) are repeated more than once.

5. The method of claim 1, wherein the first or second DNA substrate molecule comprises a gene cluster.

6. The method of claim 1, wherein at least one restriction endonuclease fragment from a DNA substrate molecule is isolated and subjected to mutagenesis to generate a library of mutant fragments.

7. The method of step 6, wherein the library of mutant fragments is used in the ligation of (b).

8. The method of claim 7, wherein the DNA substrate molecule encodes all or part of a protein selected from Table I or alpha interferon.

9. The method of claim 6, wherein mutagenesis comprises recursive sequence recombination.

10. The method of claim 1, wherein the product of (d) is subjected to mutagenesis.

11. The method of claim 10, wherein mutagenesis comprises recursive sequence recombination.

12. The method of claim 1, wherein a product of (e) is used as a DNA substrate molecule in (b).

13. The method of claim 10, wherein the product of claim 10 is used in (d).

14. The method of claim 1, wherein the recombinant DNA substrate molecule of (d) comprises a library of recombinant DNA substrate molecules.

15. A recombinant DNA molecule produced by the method of claim 1.

16. A method for evolving a protein encoded by a recombinant DNA substrate molecule by recombining at least a first and second DNA substrate molecule, the method comprising:

(a) providing at least first and second substrate molecules which differ from each other in at least one nucleotide and which comprise defined segments, the first and second substrate molecule each encoding a protein, or being homologous to a protein-coding DNA, and providing a set of oligonucleotide PCR primers, the set of PCR primers comprising a plurality of primers, each of the plurality of PCR primers comprising a first subsequence which is complementary to a first segment from the first substrate molecule and a second subsequence which is complementary to a second segment from the second substrate nucleic acid, wherein the first segment from the first substrate molecule comprises at least one nucleotide difference as compared to the second segment;

(b) amplifying the segments of the at least a first and second DNA substrate molecules with the primers of step (a) in a polymerase chain reaction;

(c) assembling the products of step (b) to generate a library of recombinant DNA substrate molecules;

(d) screening or selecting the products of (c) for a desired property; and

(e) recovering a recombinant DNA substrate molecule from (d) thereby providing a recombinant DNA substrate molecule encoding an evolved protein;

(f) expressing the evolved protein, thereby producing the evolved protein; and,

(g) subjecting the at least a first and second second DNA substrate molecules to mutagenesis prior to step (a).

17. The method of claim 16, wherein the at least a first and second DNA substrate molecules comprise alleles of a gene.

18. The method of claim 16, wherein the at least a first and second DNA substrate molecules comprise a library of mutants.

19. The method of claim 16, wherein the segments are defined by sites within intergenic regions.

20. The method of claim 16, wherein the segments are defined by sites within introns.

21. The method of claim 16, wherein the primers comprise a uracil substitution at one or more thymidine residues.

22. The method of claim 21, wherein the products of (b) are treated with uracil glycosylase.

23. The method of claim 16, wherein (a)-(e) are repeated.

24. The method of claim 16, wherein the at least a first and second DNA substrate molecule comprises a gene cluster.

25. The method of claim 16, wherein the at least first and second DNA substrate molecule encodes all or part of a DNA polymerase or alpha interferon.

26. The method of claim 16, wherein at least one PCR primer differs from the at least a first and second DNA substrate molecules in at least one nucleotide.

27. The method of claim 26, wherein the PCR primer comprises a nucleotide sequence of a known mutant or polymorphism of the at least a first or second DNA substrate molecule.

28. The method of claim 27, wherein the PCR primer is degenerate and encodes the nucleotide sequences of more than one known mutant or polymorphism of the at least a first or second DNA substrate molecule.

29. The method of claim 28, wherein the at least a first and second DNA substrate molecule encodes all or part of a protein selected from Table I or alpha interferon.

30. The method of claim 16, wherein mutagenesis comprises recursive sequence recombination.

31. The method of claim 16, wherein the products of (e) are subjected to mutagenesis.

32. The method of claim 31, wherein mutagenesis comprises recursive sequence recombination.

33. The method of claim 31, wherein the products of claim 31 are used in (b).

34. The method of claim 16, wherein the products of (e) are used as a DNA substrate molecule in (b).

35. The method of claim 16, wherein the recombinant DNA substrate molecule of (e) comprises a library of recombinant DNA substrate molecules.

36. The evolved protein or recombinant DNA substrate molecule encoding the evolved protein produced by the method of claim 16.

37. A method for evolving a protein encoded by a recombinant DNA substrate molecule, by recombining at least a first and second DNA substrate molecule, the method comprising:

(a) providing at least first and second substrate molecules, which first and second substrate molecules each encode a protein, or are homologous to a protein-coding DNA substrate molecule, which first and second substrate molecules share a region of sequence homology of about 10 to 100 base pairs and comprise defined segments and providing regions of homology in the at least a first and second DNA substrate molecules by inserting an intron sequence between at least two defined segments;

(b) fragmenting and recombining DNA substrate molecules of (a), wherein regions of homology are provided by the introns;

(c) screening or selecting the products of (b) for a desired property; and

(d) recovering the recombinant DNA substrate molecule from the products of (c), thereby providing a recombinant DNA substrate molecule encoding an evolved protein; and,

(e) expressing the evolved protein, thereby producing the evolved protein.

38. The method of claim 37, wherein the introns are self-splicing.

39. The method of claim 37, wherein the inserted introns comprise from about 1 to about 10 nonhomologous introns.

40. The method of claim 37, wherein the intron comprises a recognition site for a restriction endonucleases having non-palindromic ends at cleavage sites.

41. The method of claim 37, wherein (b)-(d) are repeated.

42. The method of claim 37, wherein the DNA substrate molecule comprises a gene cluster.

43. The method of claim 37, wherein at least one segment from a DNA substrate molecule is isolated and subjected to mutagenesis to generate a library of mutant fragments.

44. The method of claim 43, wherein the library of mutant segments is used in the recombination of (b).

45. The method of claim 37, wherein the segments are defined by exons.

46. The method of claim 37, wherein the segments are defined by intergenic regions.

47. The method of claim 37, wherein the at least a first and second DNA substrate molecules encode protein homologues.

48. The method of claim 37, wherein the intron contains a lox site, and wherein the products of (b) are used to transfect a Cre.sup.+ host.

49. The method of claim 37, wherein the at least a first and second DNA substrate molecule encodes all or part of a protein selected from Table I or alpha interferon.

50. The method of claim 37, wherein the at least a first and second DNA substrate molecule are subjected to mutagenesis prior to step (a).

51. The method of claim 50, wherein mutagenesis comprises recursive sequence recombination.

52. The method of claim 37, wherein the products of (d) are subjected to mutagenesis.

53. The method of claim 50, wherein mutagenesis comprises recursive sequence recombination.

54. The method of claim 37, wherein the products of (d) are used as a DNA substrate molecule in (b).

55. The method of claim 37, wherein the recombinant DNA substrate molecule of (d) comprises a library of recombinant DNA substrate molecules.

56. The evolved protein, or the recombinant DNA substrate molecule encoding the evolved protein produced by the method of claim 37.

57. A method for evolving a protein encoded by a DNA substrate molecule the method comprising:

(a) providing a set of oligonucleotide PCR primers, for amplification and recombination of at least a first and second DNA substrate molecule, wherein the at least a first and second substrate molecules differ from each other in at least one nucleotide and comprise defined segrnents and wherein for each junction of segments a pair of primers is provided, one member of each pair bridging the junction at one end of a segment and the other bridging the junction at the other end of the segment, with the terminal ends of the DNA molecule having as one member of the pair a generic primer, and wherein a set of primers is provided for each of the at least a first and second substrate molecules;

(b) amplifying the segments of the at least a first and second DNA substrate molecules with the primers of (a) in a polymerase chain reaction;

(c) assembling the products of (b) to generate a pool of recombinant DNA molecules;

(d) selecting or screening the products of (c) for a desired property;

(e) recovering a recombinant DNA substrate molecule from the products of (d) encoding an evolved protein; and

(f) repeating (a)-(e).

58. The method of claim 57, wherein the at least a first and second DNA substrate molecule are subjected to mutagenesis prior to (a).

59. The method of claim 57, wherein the at least a first and second DNA substrate molecule comprise sequences encoding protein homologues.

60. The method of claim 57, wherein the primers comprise a uracil substitution at one or more thymidine residues.

61. The method of claim 60, wherein the products of (b) are treated with uracil glycosylase.

62. The method of claim 57, wherein the at least a first and second DNA substrate molecule encodes all of part of a protein selected from Table I or alpha interferon.

63. The method of claim 57, wherein the at least a first and second DNA substrate molecule comprises a gene cluster.

64. An evolved protein produced by the method of claim 57.

65. The method of claim 57, wherein at least one PCR primer differs from the at least a first and second substrate molecules in at least one nucleotide.

66. The method of claim 65, wherein the PCR primer comprises a nucleotide sequence of a known mutant or polymorphism of the at least a first or second substrate molecule.

67. The method of claim 66, wherein the PCR primer comprises a nucleotide sequence of a known mutant or polymorphism of the at least a first or second substrate molecule.

68. The method of claim 58, wherein mutagenesis comprises recursive sequence recombination.

69. The method of claim 58, wherein the products of (e) are subjected to mutagenesis.

70. The method of claim 62, wherein mutagenesis comprises recursive sequence recombination.

71. The method of claim 58, wherein the products of (e) are used as a DNA substrate molecule in (b).

72. The method of claim 58, wherein the recombinant DNA substrate molecule of (e) comprises a library of recombinant DNA substrate molecules.

73. A method for recombining at least a first and second DNA substrate molecule, comprising:

(a) transfecting a host cell with at least a first and second DNA substrate molecule wherein the at least a first and second DNA substrate molecules are recombined in the host cell;

(b) screening or selecting the products of (a) for a desired property; and

(c) recovering recombinant DNA substrate molecules from (b) and

(d) repeating steps a-c.

74. The method of claim 73, wherein the products of (c) are subjected to mutagenesis.

75. The method of claim 74, wherein the mutagenesis comprises recursive sequence recombination.

76. The method of claim 73, wherein (a)-(d) are repeated.

77. The method of claim 73, wherein the products of claim 75 are used in (a).

78. The method of claim 1 further comprising expressing the evolved protein in vitro or in a cell.

79. An evolved protein produced by the method of claim 78.

80. The method of claim 1 wherein the library of step (b) comprises 10.sup.3 -10.sup.8 different members.

81. The method of claim 16 further comprising expressing the evolved protein in vitro or in a cell.

82. An evolved protein produced by the method of claim 80.

83. The method of claim 37 further comprisinig expressing the evolved protein in vitro or in a cell.

84. An evolved protein produced by the method of claim 83.

85. A method of performing oligonucleotide mediated recombination, the method comprising:

providing a first and a second nucleic acid;

selecting segments in the first and second nucleic acid;

providing a plurality of bridge oligonucleotides, which bridge oligonucleotides each comprise at least a first subsequence which is complementary to at least one segment in the first nucleic acid and at least a second subsequence which is complementary to the second nucleic acid;

extending the plurality of bridge oligonucleotides with a polymerase, using the first and second nucleic acids, or subsequences of the first and second nucleic acids, as templates, thereby producing a plurality of recombinant nucleic acid segments; and,

providing a plurality of recombinant nucleic acids, each comprising one or more subsequence comprising one or more of the recombinant nucleic acid segments.

86. The method of claim 85, wherein the plurality of recombinant nucleic acids are produced by assembly PCR performed using a pool of nucleic acids comprising the recombinant nucleic acid segments.

87. The method of claim 85, wherein the plurality of bridge oligonucleotides are extended by PCR.

88. The method of claim 85, further comprising recombining at least one of the plurality of recombinant nucleic acids with at least one additional nucleic acid to produce a further recombined nucleic acid.

89. The method of claim 88, further comprising selecting the further recombined nucleic acid for a desired property.

Details for Patent 6,335,160

Applicant Tradename Biologic Ingredient Dosage Form BLA Approval Date Patent No. Expiredate
Merck Sharp & Dohme Corp. INTRON A interferon alfa-2b For Injection 103132 06/04/1986 ⤷  Try a Trial 2015-02-17
Merck Sharp & Dohme Corp. INTRON A interferon alfa-2b For Injection 103132 ⤷  Try a Trial 2015-02-17
Merck Sharp & Dohme Corp. INTRON A interferon alfa-2b Injection 103132 ⤷  Try a Trial 2015-02-17
>Applicant >Tradename >Biologic Ingredient >Dosage Form >BLA >Approval Date >Patent No. >Expiredate

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