Methods and compositions for polypeptides engineering

• US 6,355,484 B1
• Filed: 06/24/1999
• Issued: 03/12/2002
• Est. Priority Date: 02/17/1994
• Status: Expired due to Term

• Current Assignee: Codexis, Inc.
• Original Assignee: Maxygen Inc.
• Inventors: Patten, Phillip A., Stemmer, Willem P. C.
• Primary Examiner(s): WHISENANT, ETHAN C
• Application Number:
  US09/344,002
• Time in Patent Office: 992 Days
• Field of Search: 435/440, 435/6, 435/91.2, 536/23.1, 536/24.3
• US Class Current: 435/440
• 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 making a recombined nucleic acid that encodes a product having a desired property, the method comprising:

(a) providing at least one single-stranded polynucleotide;

(b) hybridizing a plurality of nucleic acid fragments to the single-stranded polynucleotide, which nucleic acid fragments are produced by fragmentation of a plurality of non-identical substrate nucleic acids;

(c) extending and ligating the resulting hybridized nucleic acid fragments, thereby producing one or more recombined nucleic acid; and

,

(d) screening or selecting one or more product encoded by the recombined nucleic acid, or a complementary strand thereto, for the desired property, thereby identifying the recombined nucleic acid that encodes the product having the desired property.

2. The method of claim 1, wherein the single-stranded polynucleotide comprises DNA.

3. The method of claim 1, wherein the single-stranded polynucleotide is a phagemid.

4. The method of claim 1, wherein the single-stranded polynucleotide comprises uracil.

5. The method of claim 1, wherein the one or more recombined nucleic acid of products of (c) are treated with uracil glycosylase.

6. The method of claim 4, further comprising amplifying the one or more recombined nucleic acid under conditions wherein the single-stranded polynucleotide is not amplified, thereby producing a population ot recombined nucleic acids.

7. The method of claim 6, wherein the one or more recombined nucleic acid is amplified in vivo.

8. The method of claim 1, wherein the substrate nucleic acids are fragmented by sonication or shearing.

9. The method of claim 1, wherein the substrate nucleic acids are fragmented by nuclease digestion.

10. The method of claim 9, wherein the substrate nucleic acids are DNA and the nuclease is a DNAse.

11. The method of claim 1, wherein the substrate nucleic acids are allelic or species variants of one another or of the single-stranded polynucleotide.

12. The method of claim 1, wherein the substrate nucleic acids are induced variants of one another or of the single-stranded polynucleotide.

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

14. The method of claim 1, wherein the nucleic acid fragments are random fragments of the substrate nucleic acids.

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

16. The method of claim 15, wherein the nucleic acid fragments are from 10 bp-1000 bp in size.

17. The method of claim 16, wherein the nucleic acid fragments are from 20 bp--500 bp in size.

18. The method of claim 1, wherein the extending step comprises contacting the resulting hybridized nucleic acid fragments with a polymerase.

19. The method of claim 18, wherein the polymerase comprises a high-temperature polymerase.

20. The method of claim 19, wherein the high-temperature polymerase is a taq polymerase.

21. The method of claim 18, wherein the polymerase comprises a low-temperature polymerase.

22. The method of claim 21, wherein the low temperature polymerase is a Klenow polymerase.

23. The method of claim 1, wherein the nucleic acid fragments are provided in double-stranded form and denatured prior to hybridizing to the single-stranded polynucleotide.

24. The method of claim 1, wherein step (d) comprises:

(i) introducing the recombined nucleic acid, or the complementary strand thereto, into a population of cells;

(ii) expressing the recombined nucleic acid, or the complementary strand thereto, in the population of cells, thereby producing the one or more product; and

,

(iii) selecting or screening the cell population or the one or more product for the desired property.

25. The method of claim 1, wherein the single-stranded polynucleotide, or the complementary strand thereto, encodes a protein and wherein said substrate nucleic acids encode at least one additional variant of the protein.

26. The method of claim 25, wherein the protein is an enzyme and wherein the desired property is an enzymatic activity.

27. The method of claim 25, wherein the single-stranded polynucleotide encodes a wild-type protein and wherein the substrate nucleic acids encode at least one mutant form of the protein.

28. A method of identifying a recombined polynucleotide with a desired functional property, comprising:

(a) providing at least one single-stranded uracil-containing polynucleotide;

(b) providing a plurality of non-identical nucleic acid fragments capable of hybridizing to the single-stranded uracil-containing polynucleotide, wherein said plurality of nucleic acid fragments are produced by fragmentation of one or more substrate nucleic acids differing in sequence from the single-stranded uracil-containing polynucleotide;

(c) contacting the single-stranded uracil-containing polynucleotide with the plurality of nucleic acid fragments, thereby producing an annealed nucleic acid;

(d) incubating the annealed nucleic acid with a polymerase and a ligase, thereby producing a recombined polynucleotide strand annealed to the uracil-containing polynucleotide;

(e) amplifying the recombined polynucleotide strand under conditions wherein the uracil-containing polynucleotide is not amplified, thereby producing a population of recombined polynucleotides; and

,

(f) screening or selecting the population of recombined polynucleotides for the desired functional property, thereby identifying one or more polynucleotide(s) with the desired functional property.

29. The method of claim 28, wherein the single-stranded polynucleotide is DNA and fragmentation is by digestion with DNAse I.

30. The method of claim 28, wherein the one or more substrate nucleic acids are species variants of the single-stranded polynucleotide.

31. The method of claim 28, wherein the single-stranded polynucleotide is a phagemid.

32. The method of claim 28, wherein the recombined polynucleotide strand is amplified in vivo.

33. The method of claim 28, wherein step (f) comprises:

(i) introducing the recombined polynucleotides into a population of cells;

(ii) expressing the recombined polynucleotides in the population of cells; and

,

(iii) selecting or screening the cell population or the expressed protein products for the desired functional property.

34. The method of claim 28, wherein the fragments are provided in single-stranded form.

35. The method of claim 28, wherein the fragments are provided in double-stranded form and denatured prior to contacting the single-stranded polynucleotide.

36. The method of claim 28, wherein the fragments are random fragments of the one or more nucleic acids.

37. The method of claim 28, wherein the polymerase comprises a high-temperature polymerase.

38. The method of claim 37, wherein the high-temperature polymerase is a taq polymerase.

39. The method of claim 28, wherein the polymerase comprises a low-temperature polymerase.

40. The method of claim 39, wherein the low temperature polymerase is a Klenow polymerase.

41. The method of claim 28, wherein the fragments are from 5 bp to 5 kb in size.

42. The method of claim 41, wherein the fragments are from 10 bp-1000 bp in size.

43. The method of claim 42, wherein the fragments are from 20 bp-500 bp in size.

44. The method of claim 28, wherein the single-stranded polynucleotide, or a complementary strand thereto, encodes a protein and wherein said one or more substrate nuclcic acids encode at least one additional variant of the protein.

45. The method of claim 44, wherein the protein is an enzyme and wherein the desired functional property is an enzymatic activity encoded by the one or more polynucleotides.

46. The method of claim 44, wherein the single-stranded polynucleotide encodes a wild-type protein and wherein the one or more substrate nucleic acids encode at least one mutant form of the protein.

47. A method of identifying a recombined DNA molecule encoding a protein with a desired functional property, comprising:

(a) providing at least one single-stranded uracil-containing DNA molecule, which single-stranded uracil-containing DNA molecule, or a complementary strand thereto, encodes a protein;

(b) providing a plurality of non-identical DNA fragments capable of hybridizing to the single-stranded uracil-containing DNA molecule, wherein said DNA fragments are produced by fragmentation of one or more substrate DNA molecules encoding at least one additional variant of the protein and wherein the fragmentation is by digestion with DNAse I;

(c) contacting the single-stranded uracil-containing DNA molecule with the plurality of DNA fragments, thereby producing an annealed DNA molecule;

(d) incubating the annealed DNA molecule with a polymerase and a ligase, thereby producing a recombined DNA strand annealed to the uracil-containing DNA molecule;

(e) amplifying the recombined DNA strand under conditions wherein the uracil-containing DNA molecule is not amplified, thereby producing a population of recombined DNA molecules; and

,

(f) screening or selecting the population of recombined DNA molecules to identify those that encode a polypeptide having the desired functional property, thereby identifying one or more DNA molecules(s) that encode a polypeptide with the desired functional property.

48. The method of claim 47, wherein the one or more substrate DNA molecules are species variants of the single-stranded DNA molecule.

49. The method of claim 47, wherein the single-stranded DNA molecule is a phagemid.

50. The method of claim 47, wherein the recombined DNA strand is amplified in vivo.

51. The method of claim 47, wherein the DNA fragments are random fragments of the one or more substrate DNA molecules.

52. The method of claim 47, wherein the protein is an enzyme and wherein the desired functional property is an enzymatic activity.

53. The method of claim 47, wherein the single-stranded DNA molecule encodes a wild-type protein and wherein the one or more substrate DNA molecules encode at least one mutant form of the protein.

54. The method of claim 47, wherein step (f) comprises:

(i) introducing the recombined DNA molecules into a population of cells;

(ii) expressing the recombined DNA molecules in the population of cells; and

(iii) selecting or screening the cell population or the expressed protein products for the desired functional property.

55. The method of claim 47, wherein the DNA fragments are provided in single-stranded form.

56. The method of claim 47, wherein the DNA fragments are provided in double-stranded form and denatured prior to contacting the single-stranded DNA molecule.

57. The method of claim 47, wherein the polymerase comprises a high-temperature polymerase.

58. The method of claim 57, wherein the high-temperature polymerase is a taq polymerase.

59. The method of claim 47, wherein the polymerase comprises a low-temperature polymerase.

60. The method of claim 59, wherein the low-temperature polymerase is a Klenow polymerase.

61. The method of claim 47, wherein the DNA fragments are from 5 bp to 5 kb in size.

62. The method of claim 61, wherein the DNA fragments are from 10 bp-1000 bp in size.

63. The method of claim 62, wherein the DNA fragments are from 20 bp-500 bp in size.