Methods and compositions for polypeptide engineering

• US 6,319,713 B1
• Filed: 06/25/1999
• Issued: 11/20/2001
• 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:
  US09/339,904
• Time in Patent Office: 879 Days
• Field of Search: 435/6, 435/440, 536/23.1, 536/24.3, 935/76, 935/77, 935/78
• 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 for making recombined nucleic acids, the method comprising:

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

(b) providing one or more nucleic acids, at least one of which differs from the single-stranded polynucleotide(s) in at least one nucleotide, and fragmenting the one or more nucleic acids to produce a plurality of non-identical nucleic acid fragments that are capable of hybridizing to the single-stranded polynucleotide(s);

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

(d) contacting the products of (c) with a polymerase; and

,

(e) contacting the products of (d) with a ligase, thereby producing recombined nucleic acids annealed to the single-stranded polynucleotide(s).

2. The method of claim 1, wherein the nucleic acid fragments of (b) are produced by sonication or shearing of the one or more nucleic acids.

3. The method of claim 1, wherein the nucleic acid fragments of (b) are produced by fragmentation of the one or more nucleic acids using enzymatic digestion.

4. The method of claim 3, wherein the enzymatic digestion is a nuclease digestion.

5. The method of claim 4, wherein the one or more nucleic acids comprises DNA and the nuclease is a DNAse.

6. The method of claim 1, wherein said one or more nucleic acids comprises nucleic acids that are allelic or species variants of one another or of the single-stranded polynucleotide(s).

7. The method of claim 1, wherein said one or more nucleic acids comprise nucleic acids that are induced variants of one another or of the single-stranded polynucleotide(s).

8. The method of claim 1, wherein said one or more nucleic acids or said single stranded polynucleotide(s) is selected from a group consisting of:

a genomic nucleic acid, a cDNA, a vector, or a phagemid.

9. The method of claim 1 wherein the plurality of nucleic acid fragments comprises single-stranded nucleic acid fragments.

10. The method of claims 1, wherein the plurality of nucleic acid fragments comprises double-stranded nucleic acid fragments.

11. The method of claim 10, wherein the double-stranded nucleic acid fragments are denatured prior to contacting the single-stranded polynucleotide(s).

12. The method of claim 1, further comprising degrading the single-stranded polynucleotide(s) after step (e).

13. The method of claim 3, wherein the single-stranded polynucleotide(s) comprises uracil, and the method further comprises degrading the single-stranded polynucleotide(s) after step (e).

14. The method of claim 13, wherein the single-stranded polynucleotide(s) is degraded in vivo.

15. The method of claim 13, said method further comprising synthesizing a nucleic acid strand which is complementary to one or more of the recombined nucleic acids.

16. The method of claim 1, said method further comprising:

selecting or screening the recombined nucleic acids, or an encoded product thereof for a desired property.

17. The method of claim 16, said method further comprising:

recovering or isolating the recombined nucleic acids or an encoded end product thereof having the desired property.

18. The method of claim 16, said method further comprising repeating steps (a)-(e) using the recombined nucleic acids as the one or more nucleic acids, or as the single-stranded polynucleotide(s).

19. The method of claim 1, said method further comprising:

transforming the recombined nucleic acid into a host cell.

20. The method of claim 19, wherein the host cell is a mutS host cell.

21. The method of claim 1, wherein the single-stranded polynucleotide(s) is a phagemid.

22. The method of claim 1, wherein the one or more nucleic acids comprises genomic DNA.

23. The method of claim 1, wherein the one or more nucleic acids comprises cDNA.

24. The method of claim 1, wherein the single-stranded polynucleotide(s) is selected from the group consisting of:

a vector, a phagemid, an RNA and a DNA.

25. The method of claim 1, wherein the plurality of nucleic acid fragments comprises one or more of:

100 or more members, 500 or more members, or 1000 or more members.

26. The method of claim 1, wherein at least one of the nucleic acid fragments is present in the plurality of nucleic acid fragments at a concentration of less than 1% by weight of the total population of nucleic acid fragments.

27. The method of claim 1, wherein at least one of the one or more nucleic acids comprises members which are at least 50 nucleotides in length.

28. The method of claim 1, the method further comprising mutating the recombined nucleic acids.

29. The method of claim 1, wherein at least one nucleic acid of the one or more nucleic acids comprises a sequence produced by mutagenesis of a nucleic acid sequence corresponding to the single-stranded polynucleotide(s).

30. The method of claim 1, said method further comprising:

(f) selecting or screening the resulting product of (e) for a desired property;

(g) recovering one or more recombined nucleic acid from (f); and

,

(h) repeating (a)-(f) using one or more of the recombined nucleic acids of (g) in (b), as either the one or more nucleic acid(s) of (b) or as the single-stranded polynucleotide(s) of (b), whereby a further recombined nucleic acid is produced.

31. The method of claim 16 wherein the desired property is an enzymatic activity.

32. The method of claim 31, wherein the enzymatic activist is a lipase activity.

33. The method of claim 31, wherein the enzymatic activity is a protease activity.

34. The method of claim 33, wherein the protease is a subtilisin.

35. The method of claim 16, wherein the encoded product is a pharmaceutical protein.

36. The method of claim 35, wherein the pharmaceutical protein is a receptor.

37. The method of claim 35, wherein the pharmaceutical protein is an agonist.

38. The method of claim 35, wherein the pharmaceutical protein is an antagonist.

44. The method of claim 1 wherein the one or more nucleic acids have at least 70% sequence identity to the single-stranded polynucleotide(s).

45. The method of claim 44, wherein the one or more nucleic acids have at least 90% sequence identity to the single-stranded polynucleotide(s).

46. The method of claim 5, wherein the single-stranded polynucleotide(s) comprises uracil, and the method further comprises degrading the single-stranded polynucleotide(s) after step (e).

47. The method of claim 46, wherein the single stranded polynucleotide(s) are degraded in vivo.

48. The method of claim 1, wherein the one or more nucleic acids comprise a plurality of non-identical nucleic acids.

49. The method of claim 48, wherein the plurality of nucleic acids comprise DNA and the nuclease is a DNAse.

50. The method of claim 48 wherein the single-stranded polynucleotide(s) comprises uracil, and the method further comprises degrading the single-stranded polynucleotide(s) after step (e).

51. The method of claim 50 wherein the single-stranded polynucleotide(s) are degraded in vivo.

52. The method of claim 46, the method further comprising selecting or screening the recombined nucleic acids or encoded end products thereof for a desired property, wherein the desired property is an enzymatic activity.

53. The method of claim 50, the method further comprising selecting or screening the recombined nucleic acids or encoded end products thereof for a desired property, wherein the desired property is an enzymatic activity.

39. A method for making a modified or recombinant nucleic acid, the method comprising:

(a) providing a selected single-stranded template nucleic acid;

(b) contacting the selected single-stranded template nucleic acid with a population of nucleic acid fragments, wherein the population of nucleic acid fragments comprises one or more of;

(i) nucleic acid fragments which comprise nucleic acid sequences which are homologous to the single-stranded template nucleic acid;

(ii) nucleic acid fragments resulting from digestion of at least first substrate molecules with a DNase,

(iii) nucleic acid fragments which comprise nucleic acid sequences produced by mutagenesis of a parental nucleic acid,

(iv) nucleic acid fragments comprising at least one nucleic acid sequence which is homologous to the single-stranded template nucleic acid, which sequence is present in the population at a concentration of less than 1% by weight of the total population of nucleic acid fragments,

(v) nucleic acid fragments comprising at least one hundred nucleic acid sequences which are homologous to the template, or

(vi) nucleic acid fragments comprising sequences of at least 50 nucleotides, thereby producing an annealed nucleic acid product; and

(c) contacting the annealed nucleic acid with a polymerase and a ligase, thereby producing a recombined nucleic acid strand, wherein the template nucleic acid comprises uracil and the method further comprises degrading the template nucleic acid.

40. A method for making a recombined nucleic acid, the method comprising:

(a) providing a selected single-stranded template nucleic acid;

(b) contacting the selected single-stranded template nucleic acid with a population of nucleic acid fragments, wherein the population of nucleic acid fragments comprises one or more of;

(i) nucleic acid fragments which comprise nucleic acid sequences which are homologous to the single-stranded template nucleic acid;

(ii) nucleic acid fragments resulting from digestion of at least first substrate molecules with a DNase,

(iii) nucleic acid fragments which comprise nucleic acid sequences produced by mutagenesis of a parental nucleic acid,

(iv) nucleic acid fragments comprising at least one nucleic acid sequence which is homologous to the single-stranded template nucleic acid, which sequence is present in the population at a concentration of less than 1% by weight of the total population of nucleic acid fragments,

(v) nucleic acid fragments comprising at least one hundred nucleic acid sequences which are homologous to the template, or

(vi) nucleic acid fragments comprising sequences of at least 50 nucleotides, thereby producing an annealed nucleic acid product; and

(c) contacting the annealed nucleic acid with a polymerase and a ligase, thereby producing a recombined nucleic acid strand, wherein the template nucleic acid comprises uracil and the method further comprises degrading the template nucleic acid and releasing the resulting cleaved template nucleic acid from the annealed nucleic acid.

41. The method of claim 40, wherein the template nucleic acid is degraded in vivo.

42. The method of claim 40, said method further comprising synthesizing a nucleic acid strand which is complementary to the recombined nucleic acid strand.

43. A method for making a recombined nucleic acid, the method comprising:

(a) providing a selected single-stranded template nucleic acid;

(b) contacting the selected single-stranded template nucleic acid with a population of nucleic acid fragments, wherein the population of nucleic acid fragments comprises one or more of;

(i) nucleic acid fragments which comprise nucleic acid sequences which are homologous to the single-stranded template nucleic acid;

(ii) nucleic acid fragments resulting from digestion of at least first substrate molecules with a DNase,

(iii) nucleic acid fragments which comprise nucleic acid sequences produced by mutagenesis of a parental nucleic acid,

(iv) nucleic acid fragments comprising at least one nucleic acid sequence which is homologous to the single-stranded template nucleic acid, which sequence is present in the population at a concentration of less than 1% by weight of the total population of nucleic acid fragments,

(v) nucleic acid fragments comprising at least one hundred nucleic acid sequences which are homologous to the template, or

(vi) nucleic acid fragments comprising sequences of at least 50 nucleotides, thereby producing an annealed nucleic acid product;

(c) contacting the annealed nucleic acid with a polymerase and a ligase, thereby producing a recombined nucleic acid strand; and

(d) transforming the recombined nucleic acid into a host, wherein the host is a mutS host.