Methods for generating polynucleotides having desired characteristics by iterative selection and recombination

• US 6,297,053 B1
• Filed: 02/10/2000
• Issued: 10/02/2001
• Est. Priority Date: 02/17/1994
• Status: Expired due to Term

• Current Assignee: Codexis, Inc.
• Original Assignee: Maxygen Inc.
• Inventors: Stemmer, Willem P. C.
• Primary Examiner(s): WHISENANT, ETHAN C
• Application Number:
  US09/501,698
• Time in Patent Office: 600 Days
• Field of Search: 435/440, 435/6, 536/23.1, 536/24.3, 935/76, 935/77, 935/78
• US Class Current: 506/1
• CPC Class Codes: C07K 14/43595 from coelenteratae, e.g. medusae
• CPC Class Codes: C07K 14/545 IL-1
• 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 15/1027 by DNA shuffling, e.g. RSR, STEP, RPR
• 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 9/2471 Beta-galactosidase (3.2.1.23), i.e. exo-(1-->4)-beta-D-galactanase
• CPC Class Codes: C12N 9/86 acting on amide bonds in cyclic amides, e.g. penicillinase (3.5.2)
• CPC Class Codes: C12Q 1/68 involving nucleic acids
• CPC Class Codes: C12Q 1/6811 Selection methods for production or design of target specific oligonucleotides or binding molecules
• CPC Class Codes: C12Y 302/01023 Beta-galactosidase (3.2.1.23), i.e. exo-(1-->4)-beta-D-galactanase
• CPC Class Codes: C12Y 305/02006 Beta-lactamase (3.5.2.6)
• 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

Abstract

A method for DNA reassembly after random fragmentation, and its application to mutagenesis of nucleic acid sequences by in vitro or in vivo recombination is described. In particular, a method for the production of nucleic acid fragments or polynucleotides encoding mutant proteins is described. The present invention also relates to a method of repeated cycles of mutagenesis, shuffling and selection which allow for the directed molecular evolution in vitro or in vivo of proteins.

Claims

1. A method for producing a binding protein with a desired property, the method comprising:

(i) providing a population of DNA fragments comprising at least one polynucleotide encoding all or part of a binding protein;

(ii) recursively recombining the DNA fragments in vitro or in vivo to form a pool of recombined binding protein polynucleotides, which pool of binding protein polynucleotides comprises a library of shuffled DNA sequences wherein recursively recombining the DNA fragments comprises recombining the DNA fragments to form a pool of recombined binding protein polynucleotides;

selecting the pool for the desired property, thereby providing selected binding protein polynucleotides; and

recombining the selected binding protein polynucleotides and

(iii) identifying at least one recombined binding protein polynucleotide encoding a binding protein with a desired property.

2. The method of claim 1, comprising digesting at least one polynucleotide encoding all or part of a binding protein with DNAse, thereby providing the population of DNA fragments.

3. The method of claim 1, wherein the binding protein is an antibody.

4. The method of claim 3, comprising digesting at least one polynucleotide encoding all or part of an antibody with a DNAse, thereby providing the population of DNA fragments.

5. The method of claim 3, comprising providing a population of DNA fragments comprising at least one V_H or V_L polynucleotide.

6. The method of claim 5, wherein the population of DNA fragments comprises at least one complementarity determining region (CDR) or framework region.

7. The method of claim 5, further comprising ligating or grafting the at least one V_H or V_L polynucleotide in frame with a C_H or C_L polynucleotide.

12. The method of claim 3 or 8, comprising providing at least one DNA fragment corresponding to an mRNA derived from an antibody-producing cell.

13. The method of claim 12, wherein the antibody-producing cell is derived from a spleen, a lymph node or a hybridoma.

14. The method of claim 12, wherein the antibody-producing cell is derived from an immunized mouse, an immunized human, a transgenic mouse or a hybridoma.

15. The method of claim 3 or 8, comprising providing at least one DNA fragment encoding a peptide display or an antibody display library member.

16. The method of claim 15, wherein the peptide display or the antibody display library member comprises a single chain antibody.

17. The method of claim 15, comprising providing at least one polynucleotide encoding a complementarity determining region (CDR) or a framework region.

18. The method of claim 3 or 8, comprising recombining the DNA fragments by an inverse polymerase chain reaction.

19. The method of claim 3 or 8, further comprising introducing mutations into the DNA fragments provided in step (i).

20. The method of claim 19, wherein the mutations are introduced into the DNA fragments by one or more of:

PCR amplification, error-prone PCR, mutagenic amplification, and cassette mutagenesis.

21. The method of claim 3 or 8, comprising expressing the recombined antibody polynucleotides comprising members of the library of shuffled DNA sequences in cells to identify the at least one recombined polynucleotide encoding an antibody with a desired property.

22. The method of claim 3 or 8, comprising identifying at least one recombined antibody polynucleotide encoding an antibody with an ability to bind to a predetermined epitope.

23. The method of claim 3 or 8, comprising identifying at least one recombined antibody polynucleotide encoding an antibody with an increased or decreased binding affinity for a predetermined epitope as compared to a parental antibody encoded by the DNA fragments provided in step (i).

24. The method of claim 3 or 8, comprising identifying at least one recombined antibody polynucleotide with a desired property, which desired property is selected from the group consisting of:

inhibition of binding with a compound other than the predetermined epitope, stimulation of a metabolic pathway, inhibition of a metabolic pathway, signaling activity, activity as a messenger molecule, stimulation of cellular activity, inhibition of cellular activity, altered immunogenicity and catalytic activity.

25. The method of claim 3 or 8, comprising providing at least one DNA fragment comprising a human antibody polynucleotide;

and identifying an antibody with reduced immunogenicity in a human, thereby producing a humanized antibody.

26. The method of claim 3 or 8, comprising providing at least one DNA fragment encoding a polypeptide other than an antibody;

and identifying at least one polynucleotide encoding an antibody fusion protein comprising at least one subsequence of the at least one recombined antibody polynucleotide.

8. A method for producing an antibody with a desired property, the method comprising:

(i) providing a population of homologous DNA fragments comprising at least one antibody variable region gene, thereby providing a population of variable region gene fragments;

(ii) recombining the variable region gene fragments to form a pool of recombinant variable region genes, which pool of recombinant variable region genes comprises a library of shuffled DNA sequences; and

(iii) joining at least one recombinant variable region gene produced in step (ii) with at least one additional antibody gene segment to form a recombined antibody polynucleotide encoding an antibody with a desired property.

9. The method of claim 8, further comprising identifying at least one recombined antibody polynucleotide that encodes an antibody with a desired property.

10. The method of claim 8, comprising digesting at least one polynucleotide encoding all or part of an antibody variable region with a DNAse, thereby providing the population of DNA fragments.

11. The method of claim 8, comprising providing at least one polynucleotide encoding a complementarity determining region (CDR) or a framework region.