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

• US 7,288,375 B2
• Filed: 07/18/2003
• Issued: 10/30/2007
• Est. Priority Date: 02/17/1994
• Status: Expired due to Fees

• Current Assignee: Codexis, Inc.
• Original Assignee: Maxygen Inc.
• Inventors: Stemmer, Willem P. C., Crameri, Andreas
• Primary Examiner(s): Kim; Young
• Assistant Examiner(s): WOOLWINE, SAMUEL C
• Application Number:
  US10/623,036
• Publication Number:
  US 20040241853A1
• Time in Patent Office: 1,565 Days
• Field of Search: None
• US Class Current: 435/6.14
• 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 selecting from or screening a library of recombinant proteins to identify a recombinant protein having a desired functional property, said method comprising:

a) randomly fragmenting a template double-stranded DNA into a plurality of double-stranded fragments of a desired size;

b) adding to said plurality of double-stranded fragments one or more single- or double-stranded oligonucleotides, wherein said single- or double-stranded oligonucleotides comprise areas of identity and areas of heterology to the template double-stranded DNA;

c) denaturing said plurality of double-stranded fragments and said oligonucleotides to form a population of single-stranded fragments;

d) incubating said population of single-stranded fragments from step c) with a polymerase under conditions which result in the annealing of said single-stranded fragments at said areas of identity to form pairs of annealed fragments, said areas of identity being sufficient for one member of a pair to prime replication of the other member of the pair, thereby forming mutagenized double-stranded DNA molecules;

e) repeating steps c) and d) for a desired number of cycles, wherein repeated step c) further comprises denaturing the mutagenized double-stranded DNA molecules formed in step d) in previous cycles, thereby forming a library of mutagenized double-stranded DNA molecules;

f) expressing a library of recombinant proteins encoded by said library of mutagenized double-stranded DNA molecules from step e); and

g) selecting or screening said library of recombinant proteins to identify a recombinant protein with a desired functional property.

2. The method of claim 1 wherein the concentration of a specific double-stranded fragment in the plurality of double-stranded fragments in step b) is less than 1% by weight of the total amount of DNA in the combined plurality of double-stranded fragments and single- or double-stranded oligonucleotides in step b).

3. The method of claim 1 wherein the number of different specific double-stranded fragments in step a) comprises at least about 100.

4. The method of claim 1 wherein the size of the double-stranded fragments in step a) is from about 5 bp to 5 kb.

5. The method of claim 1 wherein the size of the mutagenized double-stranded DNA molecules in the library of mutagenized double-stranded DNA molecules in step e) is from about 50 bp to 100 kb.

6. The method of claim 1 wherein the template double-stranded DNA encodes a wild-type protein.

7. The method of claim 1, wherein the polymerase is Taq.

8. The method of claim 1, wherein the polymerase is Klenow polymerase.

9. The method of claim 1, wherein the template double-stranded DNA is from 50 bp to 50 kb.

10. The method of claim 1 wherein the size of the double-stranded fragments in step a) is from about 10 bp to 1000 bp.

11. The method of claim 1 wherein the size of the double-stranded fragments in step a) is from about 20 bp to 500 bp.

12. The method of claim 1, wherein the template double-stranded DNA in step a) is obtained from DNA associated with a displayed antibody that has been screened for affinity for binding a predetermined ligand, and wherein the library of recombinant proteins in step f) is expressed as an antibody display library.