Methods and compositions for polypeptide engineering

• US 6,579,678 B1
• Filed: 10/20/2000
• Issued: 06/17/2003
• Est. Priority Date: 12/18/1996
• Status: Expired due to Fees

• Current Assignee: Codexis, Inc.
• Original Assignee: Maxygen Inc.
• Inventors: Patten, Phillip A., Stemmer, Willem P. C.
• Primary Examiner(s): ZITOMER, STEPHANIE W
• Application Number:
  US09/693,350
• Time in Patent Office: 970 Days
• Field of Search: 435/6, 435/91.2, 435/69.1, 530/300, 530/350, 530/389.1, 536/23.1
• US Class Current: 435/6.14
• 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 identifying one or more evolved polynucleotides with a desired property, the method comprising:

(a) identifying one or more regions of sequence variance in a population of homologous nucleic acid sequences;

(b) providing a plurality of overlapping nucleic acid subsequences of the homologous nucleic acid sequences, which plurality of overlapping nucleic acid subsequences comprises at least one set of degenerate nucleic acids encoding the sequence variance identified in step (a), wherein the overlapping nucleic acid subsequences are produced by a polymerase chain reaction;

(c) recombining the plurality of overlapping nucleic acid subsequences of step (b) to produce a plurality of recombined nucleic acids; and

,

(d) screening or selecting the plurality of recombined nucleic acids to identify one or more evolved polynucleotides with the desired property.

2. The method of claim 1, wherein the one or more regions of sequence variance correspond to one or more codons.

3. The method of claim 1, wherein the one or more regions of sequence variance comprise one or more point mutations.

4. The method of claim 1, wherein the one or more regions of squence variance are non-disruptive of structure or function of polypeptides encoded by members of the population of homologous nucleic acid sequences.

5. The method of claim 1, wherein the homologous nucleic acid sequences comprise sequence information corresponding to homologous nucleic acids.

6. The method of claim 1, wherein the homologous nucleic acid sequences comprise RNA sequences or DNA sequences.

7. The method of claim 1, wherein the homologous nucleic acid sequences each encode a polypeptide, or are homologous to a polypeptide-encoding nucleic acid sequence.

8. The method of claim 1, wherein the homologous nucleic acid sequences are members of a gene family.

9. The method of claim 1, wherein the homologous nucleic acid sequences comprise single-stranded nucleic acid sequences.

10. The method of claim 1, wherein the homologous nucleic acid sequences comprise allelic variants of one another.

11. The method of claim 1, wherein at least one of the homologous nucleic acid sequences is a mutagenized variant.

12. The method of claim 1, wherein the homologous nucleic acid sequences each comprises a gene cluster sequence.

13. The method of claim 1, wherein the homologous nucleic acid sequences encode all or part of a polypeptide selected from Table I or alpha interferon.

14. The method of claim 1, wherein the homologous nucleic acid sequences comprise at least about 50% sequence identity.

15. The method of claim 1, wherein step (a) comprises using computer modeling tools to model variances between members of the population of homologous nucleic acid sequences or between amino acid sequences encoded by the homologous nucleic acid sequences.

16. The method of claim 1, wherein step (c) comprises recombining the plurality of overlapping nucleic acid subsequences using reassembly PCR.

17. The method of claim 1, wherein step (c) comprises recombining the plurality of overlapping nucleic acid subsequences using a polymerase.

18. The method of claim 1, wherein the plurality of overlapping nucleic acid subsequences is provided in single-stranded form.

19. The method of claim 1, wherein members of the plurality of overlapping nucleic acid subsequences comprise defined segments of the homologous nucleic acid sequences.

20. The method of claim 1, wherein regions of overlap between members of the plurality of overlapping nucleic acid subsequences comprises about 10 to about 100 nucleotides in length.

21. The method of claim 1, wherein the degenerate nucleic acids each comprise one or more degenerate codons.

22. The method of claim 1, wherein in step (c) the plurality of overlapping nucleic acid subsequences is recombined in vitro.

23. The method of claim 1, wherein in step (c) the plurality of overlapping nucleic acid subsequences is recombined in vivo.

24. The method of claim 1, wherein members of the plurality of recombined nucleic acids comprise full length genes.

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

(i) introducing the recombined nucleic acids into a cell population;

(ii) expressing the recombined nucleic acids in the cell population to produce expressed polypeptides, and,

(iii) selecting or screening one or more members of the cell population or the expressed polypeptides to identify the one or more evolved polynucleotides comprising the desired property.

26. The plurality of overlapping nucleic acid subsequences provided in step (b) of claim 1.

27. The method of claim 1, further comprising:

(e) sequencing the one or more evolved polynucleotides of step (d) to produce one or more evolved polynucleotide sequences; and

,

(f) repeating steps (a)-(d) using the one or more evolved polynucleotide sequences of step (e) as at least one source of sequence variation to be included in the degenerate nucleic acids provided in step (b).

28. The method of claim 1, wherein step (a) comprises:

(i) aligning the homologous nucleic acid sequences to identify the one or more regions of sequence variance; and

,

(ii) segmenting the homologous nucleic acid sequences into the overlapping nucleic acid subsequences.

29. The method of claim 28, wherein (ii) comprises segmenting the homologous nucleic acid sequences using at least one algorithm.

30. The method of claim 28, wherein the overlapping nucleic acid subsequences comprise defined segments of the homologous nucleic acid sequences.

31. The method of claim 1, wherein at least one member of the plurality of overlapping nucleic acid subsequences corresponds to an exon of at least one of the homologous nucleic acid sequences.

32. The method of claim 31, wherein the exon encodes a structural element of a polypeptide selected from the group consisting of:

loops, alpha helices, subdomains, whole domains, and hydrophobic cores.

33. The method of claim 1, wherein the one or more evolved polynucleotides of step (d) are subjected to mutagenesis to provide one or more mutagenized polynucleotides.

34. The method of claim 33, wherein the mutagenesis comprises recursive sequence recombination.

35. The method of claim 1, wherein the homologous nucleic acid sequences comprise species variants of one another.

36. The method of claim 1, wherein step (c) comprises recombining the plurality of overlapping nucleic acid subsequences using a ligase.

37. The method of claim 1, wherein step (c) comprises recombining the plurality of overlapping nucleic acid subsequences using a polymerase and a ligase.

38. The method of claim 14, wherein the homologous nucleic acid sequences comprise at least about 70% sequence identity.

39. The method of claim 38, wherein the homologous nucleic acid sequences comprise at least about 80% sequence identity.

40. The method of claim 39, wherein the homologous nucleic acid sequences comprise at least about 90% sequence identity.

41. The method of claim 33, wherein the mutagenesis comprises PCR mutagenesis.

42. The method of claim 33, wherein the mutagenesis comprises oligonucleotide-directed mutagenesis.

43. The method of claim 33, wherein the mutagenesis comprises site-directed mutagenesis.