Method for creating polynucleotide and polypeptide sequences

• US 7,790,381 B2
• Filed: 12/07/2006
• Issued: 09/07/2010
• Est. Priority Date: 12/08/1997
• Status: Expired due to Fees

• Current Assignee: California Institute of Technology
• Original Assignee: California Institute of Technology
• Inventors: Volkov, Alexander, Arnold, Frances, Shao, Zhixin
• Primary Examiner(s): WHISENANT, ETHAN C
• Application Number:
  US11/636,421
• Publication Number:
  US 20080268505A1
• Time in Patent Office: 1,370 Days
• Field of Search: 435/6, 435/440, 536/22.1, 536/23.1, 536/24.3
• US Class Current: 435/6.12
• CPC Class Codes: C07K 14/43595 from coelenteratae, e.g. medusae
• 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 15/1058 Directional evolution of libraries, e.g. evolution of libraries is achieved by mutagenesis and screening or selection of mixed population of organisms

Abstract

The invention provides methods for evolving a polynucleotide toward acquisition of a desired property. Such methods entail incubating a population of parental polynucleotide variants under conditions to generate annealed polynucleotides comprising heteroduplexes. The heteroduplexes are then exposed to a cellular DNA repair system to convert the heteroduplexes to parental polynucleotide variants or recombined polynucleotide variants. The resulting polynucleotides are then screened or selected for the desired property.

Claims

1. A method for evolving a polynucleotide toward acquisition of a desired property, comprising

(a) incubating a population of parental polynucleotide variants comprising allelic or species variants of a gene, or mutants created by error-prone PCR, under conditions to generate annealed polynucleotides comprising heteroduplexes;

(b) exposing the heteroduplexes to enzymes of a DNA repair system to convert the heteroduplexes to parental polynucleotide variants or recombined polynucleotide variants;

(c) screening or selecting the recombined polynucleotide variants for the desired property.

2. The method of claim 1, wherein the heteroduplexes are exposed to the enzymes of the DNA repair system in vitro.

3. The method of claim 2, wherein the enzymes of the DNA repair system are provided as cellular extracts.

4. The method of claim 1, further comprising introducing the heteroduplexes into cells, whereby the heteroduplexes are exposed to the enzymes of the DNA repair system of the cells in vivo.

5. The method of claim 4, wherein the annealed polynucleotides further comprise homoduplexes and the introducing step selects for transformed cells comprising the heteroduplexes relative to transformed cells comprising homoduplexes.

6. The method of claim 4, wherein a first polynucleotide variant is provided as a component of a first vector, and a second polynucleotide variant is provided as a component of a second vector, and the method further comprises converting the first and second vectors to linearized forms in which the first and second polynucleotide variants occur at opposite ends, whereby in the incubating step single-stranded forms of the first linearized vector reanneal with each other to form linear first vector, single- stranded forms of the second linearized vector reanneal with each other to form linear second vector, and single-stranded linearized forms of the first and second vectors anneal with each to form a circular heteroduplex bearing a nick in each strand, and the introducing step selects for transformed cells comprising the circular heteroduplexes relative to the linear first and second vector.

7. The method of claim 6, wherein the first and second vectors are converted to linearized forms by PCR.

8. The method of claim 6, wherein the first and second vectors are converted to linearized forms by digestion with first and second restriction enzymes.

9. The method of claim 1-3 wherein the population of polynucleotide variants are provided in double stranded form, and the method further comprising converting the double stranded polynucleotides to single stranded polynucleotides before the annealing step.

10. The method of claim 9, wherein the converting step comprises:

conducting asymmetric amplification of the first and second double stranded polynucleotide variants to amplify a strand of the first polynucleotide variant, and a strand of the second polynucleotide variant, whereby the strands anneal in the incubating step to form a heteroduplex.

11. The method of claim 10, wherein the first and second double-stranded polynucleotides variants are provided in vector-free form, and the method further comprises incorporating the heteroduplex into a vector.

12. The method of claim 4 wherein the population of polynucleotides comprises first and second polynucleotides provided in double stranded form, and the method further comprises incorporating the first and second polynucleotides as components of first and second vectors, whereby the first and second polynucleotides occupy opposite ends of the first and second vectors, whereby in the incubating step single-stranded forms of the first linearized vector reanneal with each other to form linear first vector, single-stranded forms of the second linearized vector reanneal with each other to form linear second vector, and single-stranded linearized forms of the first and second vectors anneal with each to form a circular heteroduplex bearing a nick in each strand, and the introducing step selects for transformed cells comprises the circular heteroduplexes relative to the linear first and second vector.

13. The method of claim 4, further comprising sealing nicks in the heteroduplexes to form covalently-closed circular heteroduplexes before the introducing step.

14. The method of claim 11, wherein the first and second polynucleotides are obtained from chromosomal DNA.

15. The method of claim 1, further comprising repeating steps (a)-(c) whereby the incubating step in a subsequent cycle is performed on recombinant variants from a previous cycle.

16. The method of claim 1, wherein the polynucleotide variants encode a polypeptide.

17. The method of claim 1, wherein the population of polynucleotide variants comprises at least 20 variants.

18. The method of claim 1, wherein the population of polynucleotide variants are at least 10 kb in length.

19. The method of claim 1, wherein the population of polynucleotide variants comprises natural variants.

20. The method of claim 1, wherein the population of polynucleotides comprises variants generated by mutagenic PCR.

21. The method of claim 4, wherein the cells are bacterial cells.

22. The method of claim 1, further comprising at least partially demethylating the population of variant polynucleotides.

23. The method of claim 22, whether the at least partially demethylating step is performed by PCR amplification of the population of variant polynucleotides.

24. The method of claim 22, wherein the at least partially demethylating step is performed by amplification of the population of variant polynucleotides in host cells.

25. The method of claim 24, wherein the host cells are defective in a gene encoding a methylase enzyme.

26. The method of claim 1 wherein the population of variant polynucleotide variants comprises at least 5 polynucleotides having at least 90% sequence identity with one another.

27. The method of claim 1, further comprising isolating a screened recombinant variant.

28. The method of claim 27, further comprising expressing a screened recombinant variant to produce a recombinant protein.

29. The method of claim 28 further comprising formulating the recombinant protein with a carrier to form a pharmaceutical composition.

30. The method of claim 1, wherein the polynucleotide variants encode enzymes selected from the group consisting of proteases, lipases, amylases, cutinases, cellulases, amylases, oxidases, peroxidases and phytases.

31. The method of claim 1, wherein the polynucleotide variants encode a polypeptide selected from the group consisting of insulin, ACTH, glucagon, somatostatin, somatotropin, thymosin, parathyroid hormone, pigmentary hormones, somatomedin, erythropoietin, luteinizing hormone chorionic gonadotropin, hyperthalmic releasing factors, antidiuretic hormones, thyroid stimulating hormone, relaxin, interferon, thrombopoietin (TPO), and prolactin.

32. The method of claim 1, wherein the polynucleotide variants encode a plurality of enzymes forming a metabolic pathway.

33. The method of claim 1, wherein the polynucleotide variants are in concatemeric form.

34. A method for evolving a polynucleotide toward acquisition of a desired property, comprising

(a) incubating a population of parental polynucleotide variants, under conditions to generate annealed polynucleotides comprising heteroduplexes;

(b) exposing the heteroduplexes to enzymes of a DNA repair system to convert the heteroduplexes to parental polynucleotide variants or recombined polynucleotide variants;

(c) screening or selecting the recombined polynucleotide variants for the desired property; and

(d) recombining screened or selected recombined polynucleotides with each other or other substrates, followed by screening or selection.