Methods and compositions for polypeptide engineering

• US 6,455,253 B1
• Filed: 04/27/2000
• Issued: 09/24/2002
• 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:
  US09/559,565
• Time in Patent Office: 880 Days
• Field of Search: 435/6, 435/91.2, 435/69.1, 435/252.3, 435/252.33, 435/463, 435/471, 435/320.1, 530/300, 530/350, 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 for evolving a polypeptide to comprise an improved serum half-life or stability, the polypeptide being encoded by a DNA substrate molecule, the method comprising:

(a) providing a library of variants of the DNA substrate molecule;

(b) transforming the variants of (a) into host cells;

(c) expressing the variants of (b) to provide polypeptide expression products;

(d) screening or selecting from the polypeptide expression products of (c) an evolved polypeptide comprising the improved serum half-life or stability relative to the polypeptide encoded by the DNA substrate molecule in vivo;

(e) recovering an evolved DNA substrate molecule that encodes the evolved polypeptide of (d); and

,

(f) expressing the evolved DNA substrate molecule, thereby providing the evolved polypeptide comprising the improved serum half-life or stability.

2. The method of claim 1, wherein the variants are generated by one or more of:

recursive sequence recombination, PCR mutagenesis, cassette mutagenesis, oligonucleotide-directed mutagenesis, site-directed mutagenesis, doped oligo mutagenesis, chemical mutagenesis, or propagation of the DNA substrate molecule through bacterial mutator strains.

3. The method of claim 1, wherein the polypeptide expression products of (c) are displayed on surfaces of the host cells.

4. The method of claim 1, further comprising repeating (a)-(e) at least once, wherein the evolved DNA substrate molecule recovered in a prior performed (e) is used as the DNA substrate molecule in a subsequent repeated (a) to further evolve the evolved DNA substrate molecule.

5. The method of claim 1, wherein the polypeptide encoded by the DNA substrate molecule comprises all or part of a protein selected from Table I or alpha interferon.

6. The evolved DNA substrate molecule produced by the method of claim 1.

7. The evolved polypeptide produced by the method of claim 1.

8. The method of claim 1, wherein the polypeptide expression products of (c) and the polypeptide encoded by the DNA substrate molecule of (d) are derivatized with a half-life extending moiety prior to determination of half-life or stability in step (d).

9. The method of claim 1, wherein the evolved polypeptide of (f) is derivitized with a half-life extending moiety.

10. The method of claims 8 or 9, wherein the half-life extending moiety is attached to an N-terminus of the polypeptide expression products, the polypeptide encoded by the DNA substrate molecule, or the evolved polypeptide.

11. The method of claim 8 or 9, wherein the half-life extending moiety is a polymer.

12. The method of claim 11, wherein the polymer is polyethylene glycol.

13. The method of claim 1, wherein the evolved DNA substrate molecule of (e) is subjected to mutagenesis to generate mutagenized products.

14. The method of claim 13, wherein the mutagenesis comprises one or more of:

recursive sequence recombination, PCR mutagenesis, cassette mutagenesis, oligonucleotide-directed mutagenesis, site-directed mutagenesis, doped oligo mutagenesis, chemical mutagenesis, or propagation of the evolved DNA substrate molecule through bacterial mutator strains.

15. The method of claim 13, wherein the mutagenized products are used as the library of variants in a repeated (a).

16. The method of claim 13, wherein at least one codon in the evolved DNA substrate molecule is substituted with a codon encoding an amino acid capable of being derivatized by a half-life extending moiety.

17. The method of claim 13, wherein at least one codon encoding an amino acid capable of being derivatized by a half-life extending moiety is added to the evolved DNA substrate molecule.

18. The method of claim 16 or 17, wherein the amino acid is lysine.

19. The method of claim 18, further comprising attaching at least one polyethylene glycol moiety to the lysine.

26. The method of claim 1, wherein (a) comprises conducting a multi-cyclic polynucleotide extension process on partially annealed polynucleotide strands having sequences from the DNA substrate molecules under conditions whereby one strand serves as a template for extension of another strand with which it is partially annealed to provide the library of variants of the DNA substrate molecules.

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

(i) providing single-stranded polynucleotides, wherein at least one of the DNA substrate molecules differs from at least one of the single-stranded polynucleotides in at least one nucleotide;

(ii) fragmenting the DNA substrate molecules to provide a mixture of fragmented substrate molecules that are capable of annealing to the single-stranded polynucleotides;

(iii) contacting the single-stranded polynucleotides with the mixture of fragmented substrate molecules to provide annealed nucleic acids; and

(iv) contacting the annealed nucleic acids with a polymerase, a ligase, or both a polymerase and a ligase to provide the library of variants of the DNA substrate molecules.

20. A method for evolving a DNA substrate molecule encoding a polypeptide comprising an improved serum half-life or stability, the method comprising:

(a) transfecting a host cell with at least first and second DNA substrate molecules wherein the first and second DNA substrate molecules are recombined in the host cell;

(b) expressing the recombined DNA substrate molecules of (a) to provide polypeptide expression products;

(c) screening or selecting from the polypeptide expression products of (b) an evolved polypeptide comprising the improved serum half-life or stability relative to the polypeptide encoded by the DNA substrate molecule; and

,

(d) derivatizing the evolved polypeptide of (c) with a half-life extending moiety.

21. The method of claim 20, wherein the half-life extending moiety is a polymer.

22. The method of claim 21, wherein the polymer is polyethylene glycol.

23. A method for evolving a DNA substrate molecule encoding a polypeptide comprising an improved serum half-life or stability, the method comprising:

(a) transfecting a host cell with at least first and second DNA substrate molecules wherein the at least first and second DNA substrate molecules are recombined in the host cell;

(b) expressing the recombined DNA substrate molecules of (a) to provide polypeptide expression products;

(c) derivatizing the polypeptide expression products of (b) with a half-life extending moiety to provide derivatized polypeptide expression products; and

,

(d) screening or selecting from the derivatized polypeptide expression products of an evolved polypeptide comprising the improved serum half-life or stability relative to the polypeptide encoded by the DNA substrate molecule derivatized with the half-life extending moiety.

24. The method of claim 23, wherein the half-life extending moiety is a polymer.

25. The method of claim 24, wherein the polymer is polyethylene glycol.

28. A method for evolving polypeptides to comprise an improved serum half-life or stability, the polypeptides being encoded by DNA substrate molecules, the method comprising:

(a) providing a library of variants of the DNA substrate molecules;

(b) transforming the variants of (a) into host cells;

(c) expressing the variants of (b) to provide polypeptide expression products;

(d) screening or selecting from the polypeptide expression products of (c) evolved polypeptides comprising the improved serum half-life or stability relative to the polypeptides encoded by the DNA substrate molecules; and

,

(e) derivatizing the evolved polypeptides of (d) with a half-life extending moiety in order to further extend the in half-life or stability of the evolved polypeptides.

29. The method of claim 28, wherein (a) comprises conducting a multi-cyclic polynucleotide extension process on partially annealed polynucleotide strands having sequences from the DNA substrate molecules under conditions whereby one strand serves as a template for extension of another strand with which it is partially annealed to provide the library of variants of the DNA substrate molecules.

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

(i) providing single-stranded polynucleotides, wherein at least one of the DNA substrate molecules differs from at least one of the single-stranded polynucleotides in at least one nucleotide;

(ii) fragmenting the DNA substrate molecules to provide a mixture of fragmented substrate molecules that are capable of annealing to the single-stranded polynucleotides;

(iii) contacting the single-stranded polynucleotides with the mixture of fragmented substrate molecules to provide annealed nucleic acids; and

(iv) contacting the annealed nucleic acids with a polymerase, a ligase, or both a polymerase and a ligase to provide the library of variants of the DNA substrate molecules.

31. The method of claim 28, wherein the half-life extending moiety is a polymer.

32. The method of claim 31, wherein the polymer is polyethylene glycol.

33. A method for evolving polypeptides to comprise an improved serum half-life or stability, the polypeptides being encoded by DNA substrate molecules, the method comprising:

(a) providing a library of variants of the DNA substrate molecules;

(b) transforming the variants of (a) into host cells;

(c) expressing the variants of (b) to provide polypeptide expression products;

(d) derivatizing the polypeptide expression products of (c) with a half-life extending moiety to provide derivatized polypeptide expression products; and

,

(e) screening or selecting from the derivatized polypeptide expression products of (d) evolved polypeptides comprising the improve serum half-life or stability relative to the polypeptides encoded by the DNA substrate molecules derivatized with the half-life extending moiety.

34. The method of claim 33, wherein (a) comprises conducting a multi-cyclic polynucleotide extension process on partially annealed polynucleotide strands having sequences from the DNA substrate molecules under conditions whereby one strand serves as a template for extension of another strand with which it is partially annealed to provide the library of variants of the DNA substrate molecules.

35. The method of claim 33, wherein (a) comprises:

(i) providing single-stranded polynucleotides, wherein at least one of the DNA substrate molecules differs from at least one of the single-stranded polynucleotides in at least one nucleotide;

(ii) fragmenting the DNA substrate molecules to provide a mixture of fragmented substrate molecules that are capable of annealing to the single-stranded polynucleotides;

(iii) contacting the single-stranded polynucleotides with the mixture of fragmented substrate molecules to provide annealed nucleic acids; and

(iv) contacting the annealed nucleic acids with a polymerase, a ligase, or both a polymerase and a ligase to provide the library of variants of the DNA substrate molecules.

36. The method of claim 33, wherein the half-life extending moiety is a polymer.

37. The method of claim 36, wherein the polymer is polyethylene glycol.