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

• US 6,291,242 B1
• Filed: 10/02/1998
• Issued: 09/18/2001
• 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:
  US09/165,060
• Time in Patent Office: 1,082 Days
• Field of Search: 435/6, 435/440, 435/172.3, 435/91.2, 935/76, 935/77, 935/78, 536/23.1, 536/24.3
• 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 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 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: C12Q 2525/179 incorporating arbitrary or random nucleotide sequences
• 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: H10N 60/128 having three or more electrodes, e.g. transistor-like structures

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 of shuffling polynucleotides, comprising:

conducting a polynucleotide extension process on overlapping segments having sequences of a population of variants of a polynucleotide encoding a plurality of genes under conditions whereby one segment serves as a template for extension of another segment to generate a population of recombinant polynucleotides at least one of which encodes the plurality of genes; and

screening or selecting recombinant polynucleotides encoding the plurality of genes to identify a recombinant polynucleotide encoding the plurality of genes having a desired functional property conferred by the genes or their expression products.

2. The method of claim 1, wherein the plurality of genes are physically clustered in nature.

3. The method of claim 1, wherein the plurality of genes form a multicomponent pathway.

4. The method of claim 3, wherein the expression products of the genes in the multicomponent pathway produce a secondary metabolite.

5. The method of claim 4, wherein the secondary metabolite is a drug, and the screening or selecting comprises spotting samples of the secondary metabolite on a lawn of cells, and identifying a sample that results in clearing of the lawn, indicating the sample is toxic to the cells.

6. The method of claim 4, wherein the secondary metabolite is an antibiotic.

7. The method of claim 3, wherein the multicomponent pathway confers heavy metal resistance.

8. The method of claim 1, wherein the variants comprise natural variants.

9. The method of claim 1, wherein the variants comprise induced variants.

10. The method of claim 2, wherein the naturally occurring variants of a polynucleotide comprise human polynucleotides.

11. The method of claim 1, wherein the naturally occurring variants of a polynucleotide comprise bacterial polynucleotides.

12. The method of claim 1, wherein the naturally occurring variants of a polynucleotide comprise plant polynucleotides.

13. The method of claim 1, wherein the naturally occurring variants of a polynucleotide comprise animal polynucleotides.

14. The method of claim 1, wherein the naturally occurring variants of a polynucleotides comprise allelic variants of the plurality of genes.

15. The method of claim 1, wherein the naturally occurring variants of a polynucleotide comprise species variants of the plurality of genes.

16. The method of claim 1, further comprising randomly fragmenting the population of variants of a polynucleotide to generate the overlapping segments before conducting the extension process.

17. The method of claim 1, wherein the population of variants of the polynucleotide are randomly fragmented by DNaseI digestion to generate the overlapping segments.

18. The method of claim 1, wherein at least one cycle of the extension process is conducted under conditions resulting in incomplete extension products of the variants of the polynucleotide.

19. The method of claim 1, further comprising expressing the recombinant polynucleotide having the desired functional property in a host to produce expression products of the genes.

20. The method of claim 19, wherein at least some of the expression products are enzymes.

21. The method of claim 20, wherein the enzymes catalyze production of a secondary metabolite.