Methods and compositions for polypeptide engineering

• US 6,586,182 B1
• Filed: 10/20/2000
• Issued: 07/01/2003
• 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:
  US09/693,389
• Time in Patent Office: 984 Days
• Field of Search: 435/6, 435/7.1, 435/91.2, 435/69.1, 435/455, 435/471, 435/472, 435/194, 435/196, 536/23.1, 530/300, 530/350
• 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 producing nucleic acids encoding mutagenized polypeptides, the method comprising:

(a) selecting a plurality of homologous polypeptide sequences;

(b) using computer modeling tools to model variances in amino acid sequence between the homologous polypeptide sequences;

(c) selecting amino acid variations between the homologous polypeptide sequences based on the computer modeling information;

(d) providing a plurality of overlapping polynucleotides comprising subsequences of nucleic acids encoding the plurality of homologous polypeptide sequences, and wherein the plurality of polynucleotides comprises subsequences encoding the amino acid variations selected in step (c); and

,

(e) reassembling the plurality of polynucleotides into nucleic acid sequences encoding mutagenized polypeptides.

2. The method of claim 1, wherein the homologous polypeptide sequences comprise allelic or species variants of one another.

3. The method of claim 1, wherein at least one of the homologous polypeptide sequences is a mutagenized variant.

4. The method of claim 1, wherein the homologous polypeptide squences are encoded by different members of a gene family.

5. The method of claim 1, wherein the homologous polypeptide sequences of step (a) are selected from the group consisting of proteins listed it Table I or alpha interferon.

6. The method of claim 1, wherein the homologous polypeptide sequences comprise at least about 50% sequence identity, at least about 70% sequence identity, at least about 80% sequence identity, or at least about 90% sequence identity.

7. The method of claim 1, wherein the amino acid variations are non-disruptive of polypeptide structure or function.

8. The method of claim 1, wherein each, overlapping polynucleotide is single-stranded.

9. The method of claim 1, wherein the plurality of overlapping palynucleotides comprises defined segments of the nucleic acids.

10. The method of claim 1, wherein the plurality of overlapping polynucleotides is reassembled in vitro.

11. The method of claim 1, wherein the plurality of overlapping polynucleotides is reassembled in vitro.

12. The method of claim 1, wherein the overlapping polynucleotides of step (d) are produced by a polymerase chain reaction.

13. The method of claim 1, wherein regions of overlap between members of the plurality of overlapping polynucleotides comprise about 10 to about 100 nucleotides in length.

14. The plurality of overlapping polynucleotides provided in step (d) of claim 1.

15. The method of claim 1, wherein step (e) comprises rassembling the pIurality of polynucleotides using reassembly PCR.

16. The method of claim 1, wherein the nucleic acid sequences of step (a) comprise a library of nucleic acid sequences.

17. The method of claim 1, wherein the nucleic acid sequences of step (e) comprise sequences encoding full length genes.

18. The method of claim 1, wherein the subsequences encoding the ano acid vaation comprise at least one set of degenerate polynucleotides.

19. The method of claim 18, wherein the degenerate polynucleotides each comprise one or more degenerate codons.

20. The method of claim 1, further comprising fragmenting one or more of the nucleic acids encoding the plurality of homologous polypeptide sequences of step (d) to produce a set of nucleic acid fragments and wherein step (e) comprises reassembling the plurality of polynucleotides and the set of nucleic acid fragments into the nucleic acid sequences encoding the mutagenized polypeptides.

21. The method of claim 20, wherein the nucleic acids encoding the plurality of homologous polypeptide sequences are fragmented by one or more of, enzymatic digestion, DNase digestion, RNase digestion, restriction enzyme digestion, sonication, or shearing.

22. The method of claim 20, wherein each member of the set of nucleic alid fragments is from 5 bp to 5 kb in size.

23. The method of claim 1, wherein at least one member of the plurality of overlapping polynucleotides corresponds to an exon of at least one of the nucleic acids encoding the plurality of homologous polypeptide sequences.

24. The method of claim 23, 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.

25. The method of claim 1, further comprising:

(f) screening or selecting expressed polypeptides encoded by the nucleic acid sequences of step (e) for one or more desired properties.

26. The method of claim 25, wherein step (f) comprises:

(i) introducing the nucleic acid sequences into a cell population;

(ii) expressing the nucleic acid sequences in the cell population to produce one or more expressed polypeptides; and

,

(iii) selecting or screening one or more members of the cell population or the one of more expressed polypeptides for the one or more desired properties.

27. The method of claim 25, further comprising:

(g) repeating steps (d)-(f) using at least one polypeptide screened or selected in step (f) as at least one source of amino acid variation to be included in the polynucleotides provided in step (d).