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

• US 6,323,030 B1
• Filed: 01/29/1999
• Issued: 11/27/2001
• 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:
  US09/240,310
• Time in Patent Office: 1,033 Days
• Field of Search: 435/6, 435/172.3, 536/23.1, 536/24.3, 935/76, 935/77, 935/70
• 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 screening a nucleic acid library encoding enzyme variants for a desired property, the method comprising:

(a) providing a population of host cells comprising a recursively recombined nucleic acid library, wherein members of the library encode variants of an enzyme; and

,

(b) screening the nucleic acid library by sorting the population of host cells, wherein the host cells are sorted by fluorescence activated cell sorting (FACS) into a plurality of collection regions by using detection of a signal produced by conversion of a substrate to a product.

2. The method of claim 1, wherein the recursively recombined nucleic acid library is produced by a method comprising:

(a) providing nucleic acid subsequences corresponding to at least one starting nucleic acid;

(b) hybridizing the subsequence nucleic acids to each other or to one or more additional nucleic acids;

(c) elongating the resulting hybridized subsequences to produce at least one recombinant nucleic acid, wherein said elongating comprises extending the hybridized subsequences with a first polymerase, denaturing the resulting extended hybridized subsequences, re-hybridizing the resulting single-stranded extended subsequences and extending the resulting re-hybridized extended sequences with the first polymerase or with a second polymerase to produce further extended sequences, and;

(d) recombining the further extended sequences with each other or with one or more additional nucleic acids to produce one or more of the members of the recursively recombined nucleic acid sequence library.

3. The method of claim 2, wherein producing the nucleic acid subsequences comprises cleaving at least two homologous nucleic acids.

4. The method of claim 2, wherein producing the nucleic acid subsequences comprises making synthetic oligonucleotides which correspond to subsequences of the at least one starting nucleic acid or to variants thereof.

5. The method of claim 1, wherein the recursively recombined nucleic acid library is produced by providing subsequences of at least one starting nucleic acid, hybridizing at least partially single stranded forms of the resulting subsequence nucleic acids to one or more additional at least partly single stranded nucleic acids, elongating the resulting hybridized subsequences with a polymerase, fragmenting at least one of the resulting elongated nucleic acids, and repeating the hybridizing and elongating steps with fragments of the elongated nucleic acids, thereby generating at least one recursively recombined nucleic acid, which at least one recursively recombined nucleic acid is a member of the recursively recombined nucleic acid library.

6. The method of claim 5, wherein producing the nucleic acid subsequences comprises cleaving at least two homologous nucleic acids.

7. The method of claim 5, wherein producing the nucleic acid subsequences comprises making synthetic oligonucleotides which correspond to subsequences of the at least one starting nucleic acid or to variants thereof.

8. The method of claim 1, wherein the recursively recombined nucleic acid library is produced by

(a) providing a starting population of a plurality of non-identical nucleic acids;

(b) shuffling the non-identical nucleic acids thereby producing the recursively recombined sequence library.

9. The method of claim 8, wherein the shuffling is conducted in vitro.

10. The method of claim 8, wherein the shuffling is conducted in vivo.

11. The method of claim 8, wherein the shuffling comprises non-homologous recombination.

12. The method of claim 11, wherein said non-homologous recombination comprises cre/lox or flp/frt recombination systems.

13. The method of claim 8, wherein the shuffling comprises stuttering.

14. The method of claim 1, wherein the recursively recombined nucleic acid library is produced by conducting a multicyclic polynucleotide extension process on overlapping segments of a plurality of variant polynucleotides under conditions whereby one segment serves as a template for extension of another segment thereby generating a recursively recombined nucleic acid library.

15. The method of claim 1, wherein the recursively-recombined nucleic acid acid library is produced using a process comprising recombination of a plurality of polynucleotides in a host cell.

16. The method of claim 15, wherein the host cell comprises a mutator strain.

17. The method of claim 15, wherein the plurality of polynucleotides have been mutagenized.

18. The method of claim 17, wherein the plurality of polynucleotides have been mutagenized by oligonucleotide directed mutagenesis.

19. The method of claim 1, wherein the signal is detected by measuring fluorescence at more than one detection wavelengths.

20. The method of claim 1, wherein the substrate is a chromogenic substrate or a fluorogenic substrate.

21. The method of claim 1, wherein conversion of the substrate to the product is catalysed by the enzyme variants.

22. The method of claim 1, wherein the substrate diffuses into the host cells.

23. The method of claim 1, wherein the enzyme variants are expressed by the host cells and are secreted.

24. The method of claim 1, wherein the host cells are contained in fluid or gel drops and wherein the fluid or gel drops are passed across a sample detector that controls gating of the host cells into the plurality of sample collection regions.

25. The method of claim 24, wherein the fluid or gel drops individually comprise about one host cell.

26. The method of claim 24, wherein the detector is a photomultiplier or a photodiode.