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

• US 6,395,547 B1
• Filed: 07/19/2000
• Issued: 05/28/2002
• Est. Priority Date: 02/17/1994
• Status: Expired due to Fees

• Current Assignee: Codexis, Inc.
• Original Assignee: Maxygen Inc.
• Inventors: Stemmer, Willem P. C.
• Primary Examiner(s): Whisenant, Ethan C.
• Application Number:
  US09/619,550
• Time in Patent Office: 678 Days
• Field of Search: 435/440,6 536/23.1,24.3 935/76,77,78
• US Class Current: 506/9
• 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 15/1037 Screening libraries presented on the surface of microorganisms, e.g. phage display, E. coli display
• 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: 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

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 of producing recombined polynucleotides with desired functional properties, the method comprising:

(a) conducting multiple cycles of denaturation, renaturation and incubation in the presence of a polymerase on polynucleotide strands having sequences from a plurality of variants of a polynucleotide, the polynucleotide strands being partially annealed through 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 recombined polynucleotides in concatemeric form;

(b) introducing the concatemers without monomerization into host cells; and

,

(c) selecting or screening the recombined polynucleotides for the desired functional property.

2. The method of claim 1 wherein the variants of a polynucleotide are generated by mutagenesis of the polynucleotide.

3. The method of claim 2, wherein the mutagenesis is oligonucleotide-directed mutagenesis.

4. The method of claim 3, wherein the oligonucleotide directed mutagenesis is performed using oligonucleotides having codon positions encoding variant amino acids selected from a defined subset of amino acid residues and codon positions encoding invariant amino acids.

5. The method of claim 4, wherein codon positions encoding the variant amino acids comprises the sequence NNK.

6. The method of claim 2, wherein the mutagenesis is by random mutagenesis.

7. The method of claim 2, wherein the mutagenesis is performed by error-prone PCR.

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

9. The method of claim 1, wherein the variants of a polynucleotide are of unknown sequence.

10. The method of claim 1, wherein the variant of a polynucleotide differ from the polynucleotide by point mutations.

11. The method of claim 1, further comprising recombining the selected or screened recombined polynucleotides, and screening the resulting sequences for further enhancement of the desired functional property.

12. The method of claim 1, wherein the desired functional property is a increased drug resistance, or capacity to bind to a receptor.

13. The method of claim 1, wherein the desired functional property is an enzymatic activity.

14. The method of claim 1, wherein the host cells are yeast or mammalian cells.

15. The method of claim 1, wherein the host cells are bacterial.

16. The method of claim 1, wherein the host cells are eukaryotic.

17. The method of claim 1, wherein the concatemers are introduced into the host cells by electroporation.

18. The method of claim 1, wherein the polynucleotide strands are produced by PCR amplification of the variants of the polynucleotide.

19. The method of claim 1, wherein the polynucleotide strands are produced by random cleavage of the variants of the polynucleotide.

20. The method of claim 1, wherein the random cleavage is by DNaseI digestion.

21. The method of claim 1, wherein the polynucleotide strands are produced by chemical synthesis.

22. A method of producing a library of cells, comprising:

recombining a population of nucleic acids in vitro to produce a set of nucleic acid variants;

introducing the set of nucleic acid variants into a plurality of cells; and

,

culturing the cells under conditions whereby the nucleic acid variants recombine with themselves or with one or more additional nucleic acid in the cells, thereby producing a set of cells comprising resulting recombinant nucleic acids, the set constituting the library of cells.

23. The method of claim 22, comprising recombining a population of nucleic acids in vitro to produce a concatamer comprising nucleic acid variants.

24. The method of claim 23, wherein the concatemers are introduced into the host cells by electroporation.

25. The method of claim 22, wherein the population of nucleic acids comprises a plurality of variants of a polynucleotide.

26. The method of claim 25, wherein the variants are generated by mutagenesis of the polynucleotide.

27. The method of claim 26, wherein the mutagenesis is performed by error-prone PCR.

28. The method of claim 26, wherein the mutagenesis is oligonucleotide-directed mutagenesis.

29. The method of claim 28, wherein the oligonucleotide cassette mutagenesis is performed using oligonucleotides having codon positions encoding variant amino acids selected from a defined subset of amino acid residues and codon positions encoding invariant amino acids.

30. The method of claim 29, wherein codon positions encoding the variant amino acids comprises the sequence NNK.

31. The method of claim 26, wherein the mutagenesis is by random mutagenesis.

32. The method of claim 25, wherein the variants of a polynucleotide are naturally occurring variants.

33. The method of claim 25, wherein the variants of a polynucleotide are of unknown sequence.

34. The method of claim 25, wherein the variants of a polynucleotide differ from the polynucleotide by point mutations.

35. The method of claim 22, further comprising screening or selecting the recombined nucleic acid variants for a desired functional property.

36. The method of claim 35, further comprising recombining the selected or screened recombined polynucleotides, and screening the resulting sequences for further enhancement of the desired functional property.

37. The method of claim 35, wherein the desired functional property is a increased drug resistance, or capacity to bind to a receptor.

38. The method of claim 35, wherein the desired functional property is an enzymatic activity.

39. The method of claim 22, wherein the host cells are yeast or mammalian cells.

40. The method of claim 22, wherein the host cells are bacterial.

41. The method of claim 22, wherein the host cells are eukaryotic.

42. The method of claim 22, comprising recombining the population of nucleic acids by conducting multiple cycles of denaturation, renaturation and incubation in the presence of a polymerase on polynucleotide strands having sequences from a plurality of variants of a polynucleotide, the polynucleotide strands being partially annealed through 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 recombined polynucleotides in concatemeric form.