Methods for recombining nucleic acids

• US 6,344,356 B1
• Filed: 06/08/2000
• Issued: 02/05/2002
• 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:
  US09/590,778
• Time in Patent Office: 607 Days
• Field of Search: 435/440, 435/91.2, 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 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 recombining nucleic acids, the method comprising:

producing fragments of at least two corresponding nucleic acids, wherein at least one of the at least two nucleic acids comprises a codon-altered nucleic acid;

hybridizing the fragments to form hybrid fragments;

elongating the resulting hybrid fragments by conducting a multi-cyclic polynucleotide extension process on the hybrid nucleic acids under conditions whereby one nucleic acid strand serves as a template for extension of another nucleic acid strand with which it is partially annealed, to generate a population of shuffled polynucleotides; and

selecting or screening the population of shuffled polynucleotides for one or more desired characteristics, thereby identifying at least one selected recombinant nucleic acid.

3. The method of claim 1 or 2, further comprising producing the codon-altered nucleic acid from a parental sequence by selecting codon alterations to the parental sequence that will increase similarity of the parental sequence to a second nucleic acid, wherein the second nucleic acid is one of the at least two corresponding nucleic acids.

4. The method of claim 1 or 2, wherein the at least two corresponding nucleic acids of claim 1 or the first plurality of nucleic acids of claim 2 comprise one or more different alleles of a parental nucleic acid sequence.

5. The method of claim 1 or 2, wherein the at least two corresponding nucleic acids of claim 1 or the first plurality of nucleic acids of claim 2 comprise one or more cognate sequences or species variants of a parental nucleic acid sequence.

6. The method of claim 1 or 2, wherein the at least two corresponding nucleic acids of claim 1 or the first plurality of nucleic acids of claim 2 comprise immunoglobulin gene sequences, green fluorescent protein gene sequences, LacZ alpha gene sequences, interleukin-1β

gene sequences, betalactamase gene sequences, gene sequences encoding metal detoxification proteins, or transcriptional or translational elements.

7. The method of claim 1 or 2, wherein the at least two corresponding nucleic acids of claim 1 or the first plurality of nucleic acids of claim 2 comprise sequences encoding taxonomically-related proteins, structurally-related proteins, functionally-related proteins, mutated variants of the proteins, or combinations thereof.

8. The method of claim 1 or 2, wherein the at least two corresponding nucleic acids of claim 1 or the first plurality of nucleic acids of claim 2 comprise two or more nucleic acid sequences prepared by oligonucleotide-directed mutagenesis, site-directed mutagenesis, chemical mutagenesis, cassette mutagenesis, or PCR mutagenesis.

9. The method of claim 1 or 2, wherein the codon altered nucleic acid comprises one or more silent mutations that influences protein expression level in one or more selected cell types.

10. The method of claim 1 or 2, wherein the codon altered nucleic acid comprises one or more silent mutations that influence protein folding in one or more selected cell types.

11. The method of claim 1 or 2, further comprising:

selecting codon substitutions which result in increased expression in E. coli of a recombinant protein encoded by the recombinant nucleic acid.

12. The method of claim 1 or 2, further comprising:

selecting codon substitutions which result in formation of one or more restriction sites within the recombinant nucleic acid.

13. The method of claim 12, wherein the codon altered nucleic acid comprises a silent mutation of codon AGT to codon AGC, both of which codons encode serine, to form a portion of an NheI site.

14. The method of claim 12, wherein the codon altered nucleic acid comprises a silent mutation of codon AAA to codon AAG, both of which codons encode lysine, to form a portion of a IfinDIII site.

15. The method of claim 12, wherein the codon altered nucleic acid comprises a silent mutation of codons TAC CCA to TAT CCG, which encode tyrosine-proline, to form a portion of a BspEl site.

16. The method of claim 12, wherein the codon altered nucleic acid comprises a silent mutation of codons CTC GAA to TTG GAG, which encode leucine-glutamic acid, to form a portion of a XhoI site.

17. The method of claim 12, wherein the codon altered nucleic acid comprises a silent mutation of codon TCC to AGC, both of which codons encode serine, to form a portion of a BamHI site.

18. The method of claim 12, wherein the codon altered nucleic acid comprises a silent mutation of codon TCG to TCC, both of which codons encode serine, to form a portion of a SalI site.

19. The method of claim 1 or 2, wherein at least one of the at least two corresponding nucleic acids or at least one member of the first plurality of nucleic acids comprises a random mutation, a pseudorandom mutation, a defined kernal mutation, or a combination thereof.

20. The method of claim 1 or 2, wherein at least one of the at least two corresponding nucleic acids of claim 1 or the first plurality of nucleic acids of claim 2 further comprises a stop codon.

21. The method of claim 1 or 2, wherein the at least two corresponding nucleic acids of claim 1 or the first plurality of nucleic acids of claim 2 comprise at least 15 to 20 residues of complementarity.

22. The method of claim 21, wherein the at least 15 to 20 residues of complementarity flank a region to be recombined.

23. The method of claim 22, wherein the region to be recombined comprises an oligonucleotide comprising one or more degenerate codons at one or more positions within the region to be recombined.

24. The method of claim 1 or 2, wherein the desired characteristic comprises an encoded protein;

a transcriptional element;

a sequence controlling transcription, translation, RNA processing, RNA stability, chromatin conformation, or gene expression;

a replicative element;

a protein-binding element;

or a combination thereof.

25. The method of claim 1, wherein the encoded protein comprises a receptor, a cell surface receptor, an intracellular protein, a glycoprotein, or an epitope.

26. The method of claim 1, further comprising:

hybridizing fragments of the selected recombinant nucleic acid to one or more additional nucleic acids or nucleic acid fragments; and

elongating the resulting recombinant hybridized fragments to produce at least one further recombinant nucleic acid.

28. The method of claim 1, comprising producing the fragments from the corresponding nucleic acids by DNAse digestion, chemical cleavage, mechanical shearing, or combinations thereof.

29. The method of claim 1, comprising producing the fragments from the corresponding nucleic acids by synthesizing oligonucleotides derived from the at least two corresponding nucleic acids.

30. The method of claim 1, comprising producing the fragments from the corresponding nucleic acids by PCR amplification.

32. The method of claim 1 or 31, further comprising hybridizing the fragments of claim 1 or the two or more nucleic acids of claim 31 in the presence of an additive which enhances a rate or extent of reannealing or recombination.

33. The method of claim 32, wherein the additive comprises polyethylene glycol, a cationic detergent, or a recombinogenic protein.

34. The method of claim 1 or 31, comprising elongating the hybrid fragments of claim 1 or the hybridized nucleic acids of claim 31 with a polymerase.

35. The method of claim 34, wherein the polymerase comprises a DNA polymerase, a thermostable polymerase, a proofreading polymerase;

a non-proofreading polymerase;

a proofreading-defective polymerase;

a heat-stable polymerase;

a heat-labile polymerase;

an evolved polymerase;

a Klenow fragment;

a Pyrococcus furiosus (Pfu) polymerase;

a Taq polymerase;

a bacteriophage T4 polymerase;

a bacteriophage T7 polymerase, a Thermus therinophilus (Tth) polymerase, a Thermus flavus (Tfl) polymerase, a Thermococcus litoralis (Tli, Vent) polymerase, a Pyrococcus woesei (Pwo) polymerase, a Thermotoga maritima (UltMa) polymerase, a Thermus brockianus (Thermozyme) polymerase, a Thennococcus sp. (9°

Nm) polymerase, a Pyrococcus sp. (‘

Deep Vent’

) DNA polymerase, an E. coli DNA polymerase I (native and Klenow forms), or an E. coli DNA polymerase III.

36. The method of claim 1 or 31, wherein elongating the hybrid fragments of claim 1 or the hybridized nucleic acids of claim 31 comprises:

extending the resulting hybridized fragments or hybridized nucleic acids with a first polymerase;

denaturing the resulting extended hybridized fragments or extended hybridized nucleic acids;

rehybridizing the resulting single-stranded extended fragments or single-stranded extended nucleic acids; and

extending the resulting rehybridized fragments or rehybridized nucleic acids with the first polymerase or with a second polymerase, to produce further extended fragments or further hybridized nucleic acids.

2. A method of recombining nucleic acids, the method comprising:

a) providing a first plurality of nucleic acids, wherein at least one member of the first plurality of nucleic acids comprises a codon-altered nucleic acid;

b) recombining the first plurality of nucleic acids to form at least one recombinant nucleic acid;

c) selecting or screening the at least one recombinant nucleic acid for at least one desired characteristic, thereby providing a selected recombinant nucleic acid;

d) recombining the selected recombinant nucleic acid with the first plurality of nucleic acids or a second plurality of nucleic acids, to form a further recombinant nucleic acid; and

e) selecting or screening the further recombinant nucleic acid for the at least one desired characteristic or an additional desired characteristic, thereby providing a further selected recombinant nucleic acid.

27. The method of claim 2, wherein providing the first plurality of nucleic acids comprises producing fragments from at least two corresponding nucleic acids.

31. The method of claim 2, wherein recombining the first plurality of nucleic acids or recombining the selected recombinant nucleic acid comprises:

hybridizing two or more nucleic acids to form hybridized nucleic acids; and

elongating the hybridized nucleic acids.

37. The method of claim 2, wherein recombining comprises performing sexual PCR, homologous recombination or site-specific recombination.