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

• US 6,180,406 B1
• Filed: 06/17/1998
• Issued: 01/30/2001
• Est. Priority Date: 02/17/1994
• Status: Expired due to Term

• Current Assignee: Codexis, Inc.
• Original Assignee: Maxygen Inc.
• Inventors: Stemmer, Willem P.C.
• Primary Examiner(s): Jones, W. Gary
• Assistant Examiner(s): WHISENANT, ETHAN C
• Application Number:
  US09/099,015
• Time in Patent Office: 958 Days
• Field of Search: 435/6, 435/91.2, 435/172.3, 435/440, 536/23.1, 536/24.3
• 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 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 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: C12Q 2525/179 incorporating arbitrary or random nucleotide sequences
• 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: H10N 60/128 having three or more electrodes, e.g. transistor-like structures

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 producing a polynucleotide having a desired functional property, said method comprising:

(a) providing a population of homologous polynucleotides, wherein at least two of the related polynucleotides differ from each other by the presence of at least one deletion or insertion;

(b) shuffling the homologous polynucleotides to produce a plurality of recombinant polynucleotides, and selecting or screening the population of recombinant polynucleotides to obtain at least one recombinant polynucleotide that has evolved toward the desired functional property; and

,

(c) shuffling the recombinant polynucleotide(s) produced in the previous step, and selecting or screening for at least one recombinant polynucleotide that has evolved toward the desired functional property.

2. The method of claim 1, further comprising the step of repeating step (c) from 1 to 50 times, until a recombinant polynucleotide has acquired the desired functional property.

3. The method of claim 1, wherein step (c) comprises selecting for at least one recombinant polynucleotide that has evolved toward the desired functional property.

4. The method of claim 1, wherein the related polynucleotides are produced by mutagenesis of a parental polynucleotide.

5. The method of claim 4, wherein at least one polynucleotide in the population of related polynucleotides differs from the parental polynucleotide by a deletion of at least one nucleotide.

6. The method of claim 4, wherein at least one polynucleotide in the population of related polynucleotides differs from the parental polynucleotide by an insertion.

7. The method of claim 6, wherein the insertion is of an oligonucleotide.

8. The method of claim 7, wherein a synthetic oligonucleotide is inserted.

9. The method of claim 7, wherein the oligonucleotide is inserted into the parental polynucleotide by ligation.

10. The method of claim 1 wherein the desired functional property is a binding activity.

11. The method of claim 1, wherein the related polynucleotides and the recombinant polynucleotides are double-stranded and at least one cycle of shuffling comprises:

a) treating the related or recombinant polynucleotides under conditions which provide for the cleavage of said polynucleotides into random double-stranded fragments of a desired size;

b) denaturing the double-stranded fragments into single-stranded fragments, whereby a mixture of overlapping single-stranded fragments is produced; and

,

c) incubating the resultant population of single-stranded fragments with a polymerase under conditions which result in the annealing of the single-stranded fragments at areas of similarity to form pairs of annealed fragments, said areas of similarity being sufficient for one member of a pair to prime replication of the other thereby forming recombinant polynucleotides.

12. The method of claim 11, wherein at least one cycle of shuffling comprises:

a) treating the related or recombinant polynucleotides under conditions which provide for the cleavage of said polynucleotides into random double-stranded fragments of a desired size;

b) combining the product of step (a) and one or more single or double-stranded oligonucleotides, wherein said oligonucleotides comprise an area of similarity and an area of heterology to the related or recombinant double-stranded polynucleotides;

c) denaturing the resultant mixture of double-stranded overlapping fragments and oligonucleotides into single-stranded fragments; and

,

c) incubating the resultant population of single-stranded fragments with a polymerase under conditions which result in the annealing of the single-stranded fragments at areas of identity similarity to form pairs of annealed fragments, said areas of similarity being sufficient for one member of a pair to prime replication of the other thereby forming recombinant polynucleotides.

28. The method of claim 1 wherein said population of homologous polynucleotides comprises allelic or species variants, and wherein said deletion or insertion is naturally occurring.

29. The method of claim 1 wherein at least one cycle of shuffling comprises conducting a multi-cyclic polynucleotide extension process on partially annealed polynucleotide strands comprising sequences from the plurality of homologous polynucleotides, the polynucleotide strands having regions of similarity and regions of heterology with each other and being partially annealed through the regions of similarity, under conditions whereby one strand serves as a template for extension of another strand with which it is partially annealed to generate the recombinant polynucleotides.

39. The method of any one of claims 1, 13, 14, 29, 30 and 38 wherein said recombinant polynucleotides encode for recombinant polypeptides and wherein said selecting or screening step comprises:

(a) introducing said population of recombinant polynucleotides into a population of cells

(b) expressing said recombinant polynucleotides in said population of cells; and

(c) selecting or screening said population of cells for the desired functional property.

40. The method of any one of claims 1, 13, 14, 29, 30 and 38 wherein said recombinant polynucleotides encode for recombinant polypeptides and wherein said selecting or screening step comprises:

(a) expressing said recombinant polynucleotides in vitro to produce said population of recombinant polypeptides; and

(b) selecting or screening said population of recombinant polypeptides for the desired functional property.

41. The method of any one of claims 1, 13, 14, 29, 30 and 38, wherein the recombinant polynucleotide with the desired functional property encodes an enzyme.

42. The method of any one of claims 1, 13, 14, 29, 30 and 38, wherein the recombinant polynucleotide with the desired functional property encodes a viral coat protein.

43. The method of any one of claims 1, 13, 14, 29, 30 and 38, wherein the recombinant polynucleotide with the desired functional property encodes an antibody chain.

44. The method of any one of claims 1, 13, 14, 29, 30 and 38, wherein the recombinant polynucleotide with the desired functional property encodes a polymerase.

45. The method of any one of claims 1, 13, 14, 29, 30 and 38, further comprising expressing the recombinant polynucleotide with the desired functional property to produce an expression product thereof.

46. The method of claim 1, wherein at last one shuffling step comprises generating partial length copies of the homologous polynucleotides under conditions in which at least 20% of products are partial length products, whereby the partial length products anneal in new pairings and one member of a pair primes replication of the other, thereby forming the recombinant polynucleotides.

55. The method of claim 29 wherein the partially annealed polynucleotide strands are produced by providing overlapping single-stranded segments of the plurality of homologous polynucleotides and incubating under annealing conditions whereby the single-stranded segments from the different homologous polynucleotides anneal to form the partially annealed polynucleotide strands.

67. The method of claim 29 wherein said plurality of homologous polynucleotides are species variants.

13. A method for producing a polynueleotide having a desired property, said method comprising:

conducting a multi-cycle polynucleotide extension process on overlapping segments having sequences of a population of related polynucleotides under conditions whereby one segment serves as a template for extension of another segment, to generate a population of recombinant polynucleotides, wherein said related polynucleotides differ from each other by the presence of deletions or insertions; and

screening or selecting recombinant polynucleotides or expression products thereof to identify at least one recombinant polynucleotide having a desired functional property.

48. The method of claim 13, further comprising conducting a polynucleotide extension reaction on the related polynucleotides to be shuffled under conditions in which at least 20% of products are partial length products to generate the overlapping segments.

65. The method of claim 13 wherein said population of related polynucleotides are species variants.

14. A method for producing a polynucleotide having a desired property, said method comprising:

(a) choosing a plurality of polynucleotide variants to be shuffled, wherein at least one polynucleotide variant has been mutagenized;

(b) conducting a multi-cyclic polynucleotide extension process on partially annealed polynucleotide strands comprising sequences from the plurality of polynucleotide variants, the polynucleotide strands having regions of similarity and regions of heterology with each other and being partially annealed through the regions of similarity, under conditions whereby one strand serves as a template for extension of another strand with which it is partially annealed to generate a population of recombinant polynucleotides; and

(c) selecting and screening recombinant polynucleotides to identify at least one polynucleotide having a desired functional property.

15. The method of claim 14 wherein said mutagenesis is the introduction of at least one deletion.

16. The method of claim 15 wherein said mutagenesis is the introduction of at least one insertion.

17. The method of claim 16 wherein said insertion is of an oligonucleotide.

18. The method of claim 17 wherein said oligonucleotide is a synthetic oliognucleotide.

19. The method of claim 16 wherein an intron sequence is introduced between exons in at least one of the polynucleotide variants.

20. The method of claim 19 wherein said intron sequence is a naturally-occurring intron sequence.

21. The method of claim 19 wherein said intron sequence is inserted between exons in at least two polynucleotide variants, and wherein the intron sequence promotes homologous recombination between the exons.

22. The method of claim 19 wherein a plurality of intron sequences are introduced in the polynucleotide variants.

23. The method of claim 22 wherein said plurality of intron sequences have sufficient sequence identity to promote homologous recombination between the exons separated by the intron sequences.

24. The method of claim 19 wherein said exon sequences are naturally occurring exons.

25. The method of claim 14 wherein said mutagenesis is oligonucleotide directed mutagenesis.

26. The method of claim 14 wherein said mutagenesis is error-prone PCR.

27. The method of claim 14 wherein said mutagenesis is chemical mutagenesis.

49. The method of claim 14, further comprising conducting a polynucleotide extension reaction on the polynucleotide variants to be shuffled under conditions in which at least 20% of products are partial length products to generate the partially annealed polynucleotide strands.

50. The method of claim 14 wherein the partially annealed polynucleotide strands are produced by providing overlapping single-stranded segments of the plurality of polynucleotide variants and incubating under annealing conditions whereby the single-stranded segments from the different polynucleotide variants anneal to form the partially annealed polynueleotide strands.

51. The method of claim 50 wherein the overlapping single-stranded segments are random segments of the polynucleotide variants.

52. The method of claim 50 wherein the overlapping single-stranded segments are non-random segments of the polynucleotide variants.

53. The method of claim 50 wherein the plurality of overlapping single-stranded segments are produced by cleaving the population of polynucleotide variants to produce a population of overlapping double-stranded fragments, and denaturing the double-stranded fragments to produce the overlapping single-stranded segments.

54. The method of claim 50 wherein the overlapping single-stranded segments are produced on a DNA synthesizer.

56. The method of claim 54 wherein the overlapping single-stranded segments are random segments of the homologous polynucleotides.

57. The method of claim 54 wherein the overlapping single-stranded segments are non-random segments of the homologous polynucleotides.

58. The method of claim 54 wherein the plurality of overlapping single-stranded segments are produced by cleaving the population of homologous polynucleotides to produce a population of overlapping double-stranded fragments, and denaturing the double-stranded fragments to produce the overlapping single-stranded segments.

59. The method of claim 54 wherein the overlapping single-stranded segments are produced on a DNA synthesizer.

61. The method of claim 59 wherein the overlapping single-stranded segments are random segments of the polynucleotides.

62. The method of claim 59 wherein the overlapping single-stranded segments are non-random segments of the polynucleotides.

63. The method of claim 59 wherein the plurality of overlapping single-stranded segments are produced by cleaving the population of polynucleotides to produce a population of overlapping double-stranded fragments, and denaturing the double-stranded fragments to produce the overlapping single-stranded segments.

64. The method of claim 59 wherein the overlapping single-stranded segments are produced on a DNA synthesizer.

66. The method of claim 14 wherein said plurality of polynucleotide variants are species variants.

69. The method of any one of claim 50, 55 or 60, wherein the overlapping single stranded segments are produced by PCR amplification.

30. A method of producing a polynucleotide having a desired functional property comprising:

(a) providing a plurality of polynucleotides comprising exon sequences separated by an intron sequence, at least one of which polynucleotide is in cell-free form;

(b) shuffling the population of polynucleotides to produce recombinant polynucleotides;

(c) selecting or screening for recombinant polynucleotides that have evolved toward the desired property;

(d) repeating steps

     2) and

     3) with the recombinant polynucleotides selected in step

     3) until a recombinant polynucleotide has acquired the desired functional property.

31. The method of claim 30 wherein said intron sequence is a naturally-occurring intron sequence.

32. The method of claim 30 wherein wherein the intron sequence promotes homologous recombination between the exons.

33. The method of claim 30 wherein a plurality of intron sequences are introduced.

34. The method of claim 33 wherein at least two of said plurality of intron sequences have sufficient sequence identity to promote homologous recombination between the intron sequences.

35. The method of claim 30 wherein said exon sequences are naturally occurring exons.

36. The method of claim 30 wherein at least one shuffling step is performed in vitro.

37. The method of claim 30 wherein at least one shuffling step is performed in vivo.

38. The method of claim 30 wherein at least cycle of shuffling comprises conducting a multi-cyclic polynucleotide extension process on partially annealed polynucleotide strands comprising sequences from the plurality of polynucleotides, the polynucleotide strands having regions of similarity and regions of heterology with each other and being partially annealed through the regions of similarity, under conditions whereby one strand serves as a template for extension of another strand with which it is partially annealed to generate a population of recombinant polynucleotides.

47. The method of claim 30, wherein at least one shuffling step comprises generating partial length copies of the polynucleotides to be shuffled under conditions in which at least 20% of products are partial length products, whereby the partial length products anneal in new pairings and one member of a pair primes replication of another thereby forming the recombinant polynucleotides.

60. The method of claim 38 wherein the partially annealed polynucleotide strands are produced by providing overlapping single-stranded segments of the plurality of polynucleotides and incubating under annealing conditions whereby the single-stranded segments from the different polynucleotides anneal to form the partially annealed polynucleotide strands.

68. The method of claim 30 or 38 wherein said plurality of polynucleotides are species variants.