Methods and compositions for polypeptide engineering

• US 6,653,072 B1
• Filed: 06/24/1999
• Issued: 11/25/2003
• 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:
  US09/339,926
• Time in Patent Office: 1,615 Days
• Field of Search: 536/23.1, 536/24.3, 536/24.33, 536/25.4, 435/6, 435/91.2, 435/69.1, 435/455, 435/471
• 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 for improving expression of at least one protein, wherein the at least one protein is encoded by at least one DNA substrate molecule, comprising:

(a) providing a set of oligonucleotides, wherein each oligonucleotide comprises at least two regions complementary to the at least one DNA substrate molecule and at least one degenerate region, each degenerate region comprising a nucleic acid sequence that differs in at least one nucleotide from the complement of the at least one DNA substrate molecule;

(b) assembling the set of oligonucleotides into a library of recombinant nucleic acids;

(c) expressing the library of recombinant nucleic acids of (b) in a population of host cells; and

(d) screening or selecting the population of host cells of (c) to identify at least one host cell expressing at least one protein encoded by at least one recombinant nucleic acid, wherein expression of the at least one protein encoded by the at least one recombinant nucleic acid in the at least one host cell is improved compared to expression of the at least one protein encoded by the at least one DNA substrate molecule in a host cell.

2. The method of claim 1, wherein the at least one protein comprises bovine intestinal alkaline phosphatase.

3. The method of claim 2, wherein the oligonucleotides comprise one or more primers from Table II.

4. The method of claim 1, wherein the at least one DNA substrate molecule encodes all or part of one or more proteins selected from Table I.

5. The method of claim 1, wherein the at least one DNA substrate molecule comprises at least one gene cluster, wherein the at least one gene cluster comprises at least one nucleic acid sequence encoding the at least one protein.

6. The method of claim 1, wherein each of the oligonucleotides comprises at least a 5′

and a 3′

nucleotide complementary to the at least one DNA substrate molecule and about 20×

300 nucleotides complementary to about 85% of nucleotides in at least one segment of the at least one DNA substrate molecule.

7. The method of claim 6, wherein each oligonucleotide overlaps with a second oligonucleotide from the set of oligonucleotides.

8. The method of claim 1, further comprising:

(e) recovering the at least one recombinant nucleic acid encoding the at least one protein expressed by the at least one host cell identified in (d).

9. The method of claim 8, wherein (a)-(e) are repeated.

10. The method of claim 1, wherein the at least two regions complementary to the at least one DNA substrate molecule and the at least one degenerate region each comprise about 20 nucleotides.

11. The method of claim 1, wherein the at least one degenerate region is positioned between the at least two regions complementary to the at least one DNA substrate molecule.

12. The method of claim 8, wherein the at least one recombinant nucleic acid recovered in (e) is subjected to mutagenesis.

13. The method of claim 12, wherein the mutagenesis comprises recursive sequence recombination.

14. The method of claim 8, wherein the at least one recombinant nucleic acid of (e) comprises a library of recombinant nucleic acids.

15. The at least one protein produced by the method of claim 1.

34. The method of claim 1, comprising improving the expression of at least two proteins, wherein each of said at least two proteins is encoded by a different DNA substrate molecule.

35. The method of claim 1, comprising improving the expression of at least two proteins encoded by a single DNA substrate molecule.

16. A method for improving secretion of a target protein from a host cell, comprising:

(a) providing at least two nucleic acid sequences encoding at least one protein having a secretory function or fragments of said nucleic acid sequences, wherein said at least two nucleic acid sequences, or fragments of said nucleic acid sequences, differ from each other in at least one nucleotide;

(b) recombining said at least two nucleic acid sequences or said fragments of (a) to generate a library of recombinant nucleic acid sequences;

(c) transforming a population of host cells with the library of recombinant nucleic acid sequences of (b), wherein each host cell comprises at least one recombinant nucleic acid sequence; and

(d) screening or selecting the population of host cells of (c) to identify at least one host cell expressing the target protein, wherein secretion of the target protein by the at least one host cell is improved compared to secretion of the target protein by a host cell that does not contain the at least one recombinant nucleic acid sequence contained in the identified host cell.

17. The method of claim 16, wherein at least one of the at least two nucleic acid sequences of (a) comprises a gene cluster.

18. The method of claim 16, further comprising:

(e) recovering at least one recombinant nucleic acid sequence from the at least one identified host cell.

19. The at least one recombinant nucleic acid sequence recovered by the method of claim 18.

20. The method of claim 18, wherein the at least one recombinant nucleic acid sequence recovered in (e) comprises a library of recombinant nucleic acid sequences.

21. The method of claim 18, wherein (a)-(e) are repeated at least once.

22. The method of claim 21, wherein the at least one recombinant nucleic acid sequence recovered in at least one preceding (e) is used as at least one of the at least two nucleic acid sequences in (a).

23. The method of claim 18, wherein the at least one recombinant nucleic acid sequence recovered in (e) is subjected to mutagenesis.

24. The method of claim 23, wherein the mutagenesis comprises recursive sequence recombination.

25. The method of claim 23, wherein at least one mutagenized recombinant nucleic acid sequence is used in a repeated (a) as at least one of the at least two nucleic acid sequences.

26. The method of claim 16, wherein at least one of the nucleic acid sequences of (a), or fragment thereof, encodes at least two proteins having secretory functions.

27. The method of claim 17, wherein at least one gene of the gene cluster comprises at least one recognition site for a restriction endonuclease, the at least one recognition site having nonpalindromic ends.

28. The method of claim 16, wherein the host cell is E. coli, yeast, Bacillus, Pseudomonas, or a mammalian cell.

29. The method of claim 16, wherein the target protein is a thermostable DNA polymerase.

30. The method of claim 16, wherein the target protein is inducibly expressed.

31. The method of claim 16, wherein the target protein is linked to at least one secretory leader sequence.

32. The method of claim 16, wherein the at least one recombinant nucleic acid sequence of (c) encodes all or part of at least one protein selected from Table I.

33. The method of claim 16, wherein the at least one recombinant nucleic acid sequence of (c) comprises a library of mutant sequences.