Directed evolution method

• US 20040005594A1
• Filed: 03/13/2003
• Published: 01/08/2004
• Est. Priority Date: 09/13/2000
• Status: Active Grant

• Current Assignee: UK Research and Innovation
• Original Assignee: Medical Research Council (Government of United Kingdom)
• Inventors: Holliger, Phillipp, Ghadessy, Farid, Ong, Jennifer Lee
• Granted Patent: US 7,514,210 B2
• Time in Patent Office: Days
• US Class Current: 435/6
• CPC Class Codes: C07K 2319/00 Fusion polypeptide
• 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 15/1075 by coupling phenotype to genotype, not provided for in other groups of this subclass
• CPC Class Codes: C12N 9/1252 DNA-directed DNA polymerase (2.7.7.7), i.e. DNA replicase
• CPC Class Codes: C12Q 1/6867 Replicase-based amplification, e.g. using Q-beta replicase

Abstract

We describe a method of selecting an enzyme having replicase activity, the method comprising the steps of: (a) providing a pool of nucleic acids comprising members each encoding a replicase or a variant of the replicase; (b) subdividing the pool of nucleic acids into compartments, such that each compartment comprises a nucleic acid member of the pool together with the replicase or variant encoded by the nucleic acid member; (c) allowing nucleic acid replication to occur; and (d) detecting amplification of the nucleic acid member by the replicase. Methods for selecting agents capable of modulating replicase activity, and for selecting interacting polypeptides are also disclosed.

Claims

1. A method of selecting a nucleic acid processing (nap) enzyme, the method comprising the steps of:

(a) providing a pool of nucleic acids comprising members encoding a NAP enzyme or a variant of a NAP enzyme;

(b) subdividing the pool of nucleic acids into compartments, such that each compartment comprises a nucleic acid member of the pool together with the NAP enzyme or variant encoded by the nucleic acid member;

(c) allowing nucleic acid processing to occur; and

(d) detecting processing of the nucleic acid member by the NAP enzyme, whereby a NAP enzyme is selected.

12. The method of claim 1, wherein the NAP enzyme is a replicase enzyme that performs nucleic acid replication.

13. The method of claim 1, wherein the NAP enzyme is a replicase enzyme and the processing of the nucleic acid member constitutes replication of said member.

14. The method of claim 1, wherein the NAP enzyme is a replicase enzyme and the processing of the nucleic acid member comprises either a fill-in reaction of a 5′

overhang appended to said member or an extension of a 3′

end of said member.

15. The method of claim 12, in which amplification of the nucleic acid results from more than one round of nucleic acid replication.

16. The method of claim 15, in which the replication of the nucleic acid comprises an exponential amplification.

17. The method of claim 15, in which the amplification comprises a polymerase chain reaction (PCR), a reverse transcriptase-polymerase chain reaction (RT-PCR), a nested PCR, a ligase chain reaction (LCR), a transcription based amplification system (TAS), a self-sustaining sequence replication (3SR), NASBA, a transcription-mediated amplification reaction (TMA), or a strand-displacement amplification (SDA).

18. The method of claim 12, in which the post-amplification copy number of the nucleic acid member is substantially proportional to the activity of the replicase.

19. The method of claim 12, in which nucleic acid replication is detected by assaying the copy number of the nucleic acid member.

20. The method of claim 12, in which nucleic acid replication is detected by assaying the copy number of the nucleic acid member.

21. The method of claim 12, in which nucleic acid replication is detected by assaying the presence of tagging of the nucleic acid member.

22. The method of claim 12, in which nucleic acid replication is detected by determining the activity of a polypeptide encoded by the nucleic acid member.

23. The method of claim 1, in which the conditions in the compartment are selected to permit selection of a NAP enzyme with a particular desired property.

24. The method of claim 12, in which the replicase activity is a template-dependent replicase activity selected from a polymerase activity, a reverse transcriptase activity or a ligase activity.

25. The method of claim 1, in which the polypeptide is produced from the nucleic acid by in vitro transcription and translation.

26. The method of claims 1, wherein the polypeptide is produced from the nucleic acid in vivo in an expression host.

27. The method of claim 26 wherein said expression host is a bacterial cell.

28. The method of claim 1, in which the compartments comprise aqueous compartments of a water-in-oil emulsion.

29. The method of claim 28, in which the water-in-oil emulsion is produced by emulsifying an aqueous phase with an oil phase and a surfactant comprising Span80, Tween80, and TritonX100.

30. The method of claim 29 wherein said surfactant comprises 4.5% v/v Span80, 0.4% v/v Tween80 and 0.1% v/v TritonX100.

31. A NAP enzyme identified by a method according to claim 1.

32. The NAP enzyme of claim 31, wherein said NAP enzyme is a variant of a known NAP enzyme, and wherein said variant has greater thermostability than said known NAP enzyme.

33. The NAP enzyme of claim 31, wherein said NAP enzyme is a variant of a known NAP enzyme, and wherein said variant is inhibited to a lesser extent by heparin than is said known NAP enzyme.

34. The NAP enzyme of claim 33 which is a Taq polymerase active at a concentration of 0.083 units/μ

l or more of heparin.

35. The NAP enzyme of claim 31 which is a Taq polymerase active at a concentration of 0.083 units/μ

l or more of heparin.

36. The NAP enzyme of claim 31 which is a replicase enzyme that extends a primer having a 3′

mismatch.

37. The NAP enzyme of claim 31 which is a replicase enzyme that extends a primer having a 3′

unnatural base.

38. The NAP enzyme of claim 37 which is capable of extending a primer having a 3′

terminal base comprising 5-nitroindole or 3-carboxyamide-5-nitroindole.

39. The NAP enzyme of claim 31 which is a replicase enzyme variant of a known replicase enzyme, wherein said variant incorporates α

-thio dNTPs as nucleotide substrates more efficiently than said known replicase enzyme.

40. The NAP enzyme of claim 31 which is a replicase enzyme variant of a known replicase enzyme, wherein said variant replicates a substrate 23 kb in size in the absence of processivity factors or a 3′

-5′

exonuclease proof-reading domain.

41. The NAP enzyme of claim 36 in which the 3′

mismatch is a 3′

purine-purine mismatch or a 3′

pyrimidine-pyrimidine mismatch.

42. The NAP enzyme of claim 36 in which the 3′

mismatch is an A-G mismatch or in which the 3′

mismatch is a C-C mismatch.

43. The method of claim 1 wherein a NAP enzyme is selected that is a variant of a known enzyme and wherein said variant has greater thermostability than said known enzyme.

44. The method of claim 43 wherein the half-life of the variant at 97.5°

C. is greater than 10 times the half-life of said known enzyme at 97.5°

C.

45. The method of claim 1 wherein a polymerase enzyme is selected that is a variant of a known enzyme and wherein said variant is inhibited to a lesser extent by heparin than is said known enzyme.

2. A method of selecting an agent capable of modifying the activity of a NAP enzyme, the method comprising the steps of:

(a) providing a NAP enzyme;

(b) providing a pool of nucleic acids comprising members encoding one or more candidate agents;

(c) subdividing the pool of nucleic acids into compartments, such that each compartment comprises a nucleic acid member of the pool, the agent encoded by the nucleic acid member, and the NAP enzyme; and

(d) detecting processing of the nucleic acid member by the NAP enzyme.

3. The method according to claim 2, in which the agent is a promoter of NAP enzyme activity.

4. The method according to claim 2, in which the agent is an enzyme, preferably a kinase or a phosphorylase, which acts on the NAP enzyme to modify its activity.

5. The method according to claim 2, in which the agent is a polypeptide involved in a metabolic pathway, the pathway having as an end product a substrate which is involved in a nucleic acid processing reaction.

6. The method according to claim 2, in which the agent is a polypeptide that produces a substrate or consumes an inhibitor in a nucleic acid processing reaction.

7. A method according to claim 2, in which the agent is a polypeptide that modifies a nucleotide primer or nucleoside triphosphate substrate used in a nucleic acid processing reaction such that

a) its 3′

end becomes extendable by a NAP enzyme;

or

b) a substrate appendage added to the nucleotide primer or nucleoside triphosphate is modified by said polypeptide agent so as to allow detection or capture of a nucleotide primer or nucleoside triphosphate having the modified substrate appendage.

8. A method of selecting a pair of polypeptides that stably interact, the method comprising:

(a) providing a first nucleic acid and a second nucleic acid, the first nucleic acid encoding a first fusion protein comprising a first subdomain of a NAP enzyme fused to a first polypeptide, the second nucleic acid encoding a second fusion protein comprising a second subdomain of a NAP enzyme fused to a second polypeptide;

in which stable interaction of the first and second NAP subdomains generates processing activity, and in which at least one of the first and second nucleic acids is provided in the form of a pool of nucleic acids encoding variants of the respective first and/or second polypeptide(s);

(b) subdividing the pool or pools of nucleic acids into compartments, such that each compartment comprises a first nucleic acid and a second nucleic acid together with respective fusion proteins encoded by the first and second nucleic acids;

(c) allowing the first polypeptide to bind to the second polypeptide, such that binding of the first and second polypeptides leads to stable interaction of the NAP subdomains to generate NAP enzyme activity; and

(d) detecting processing of at least one of the first and second nucleic acids by the NAP enzyme.

9. A method of selecting a pair of polypeptides capable of stable interaction, the method comprising:

(a) providing a first nucleic acid and a second nucleic acid, the first nucleic acid encoding a first fusion protein comprising a first subdomain of polypeptide that enhances the activity of a NAP enzyme fused to a first polypeptide, the second nucleic acid encoding a second fusion protein comprising a second subdomain of polypeptide that enhances the activity of a NAP enzyme fused to a second polypeptide;

in which stable interaction of the first and second NAP subdomains generates processing activity, and in which at least one of the first and second nucleic acids is provided in the form of a pool of nucleic acids encoding variants of the respective first and/or second polypeptide(s);

(b) subdividing the pool or pools of nucleic acids into compartments, such that each compartment comprises a first nucleic acid and a second nucleic acid together with respective fusion proteins encoded by the first and second nucleic acids;

(c) allowing the first polypeptide to bind to the second polypeptide, such that binding of the first and second polypeptides leads to stable interaction of the subdomains of the NAP activity enhancing polypeptide to generate NAP enzyme activity; and

(d) detecting processing of at least one of the first and second nucleic acids by the NAP enzyme.

10. A method of selecting a polypeptide that stably folds, the method comprising:

(a) providing a pool of nucleic acids comprising members encoding one or more candidate polypeptides fused to a NAP enzyme;

(b) subdividing the pool of nucleic acids into compartments, such that each compartment comprises a nucleic acid member of the pool, the fusion polypeptide encoded by the nucleic acid member, and the NAP enzyme; and

allowing for folding of the fusion polypeptide, wherein a non-folded or improperly folded fusion polypeptide inhibits the NAP enzyme and a properly folded fusion polypeptide does not inhibit NAP activity; and

(c) detecting processing of the nucleic acid member by the NAP enzyme such that a polypeptide that stably folds is selected.

11. A method of selecting a chaperone polypeptide that promotes stable polypeptide folding, the method comprising:

(a) providing a poorly folding polypeptide fused to a NAP enzyme and a candidate chaperone;

(b) providing a pool of nucleic acids comprising members encoding one or more candidate chaperones;

(c) subdividing the pool of nucleic acids into compartments, such that each compartment comprises a nucleic acid member of the pool, the candidate chaperone encoded by the nucleic acid member, and the NAP-enzyme fusion to the poorly folding polypeptide;

(d) permitting chaperone-aided folding of the fusion polypeptide, wherein a non-folded or improperly folded fusion polypeptide inhibits the NAP enzyme and a properly folded fusion polypeptide does not inhibit NAP activity; and

(e) detecting processing of the nucleic acid member by the NAP enzyme, whereby a chaperone polypeptide is selected that promotes stable polypeptide folding.

46. A Taq polymerase mutant comprising the mutations:

F73S, R205K, K219E, M236T, E434D and A608V.

47. The Taq polymerase mutant of claim 46 that has at least four-fold increased thermostability at 97.5°

C. relative to wild-type Taq polymerease.

48. A Taq polymerase mutant comprising the mutations:

F73S, R205K, K219E, M236T, E434D and A608V.

49. The Taq polymerase mutant of claim 48 that has at least ten-fold increased thermostability at 97.5°

C. relative to wild-type Taq polymerease.

50. A Taq polymerase mutant comprising the mutations:

K225E, E388V, K540R, D578G, N583S and M747R.

51. The Taq polymerase mutant of claim 50 that has at least 100-fold increased resistance to heparin relative to wild-type Taq polymerase.

52. A Taq polymerase mutant comprising the mutations:

G84A, D144G, K314R, E520G, A608V, E742G.

53. A Taq polymerase mutant comprising the mutations:

D58G, R74P, A109T, L245R, R343G, G370D, E520G, N583S, E694K, A743P.

54. A water-in-oil emulsion comprising a surfactant comprising Span80, Tween80 and TritonX100.

55. The water-in-oil emulsion of claim 54 wherein said surfactant comprises 4.5% v/v Span80, 0.4% v/v Tween80 and 0.1% v/v TritonX100.

56. A water-in-oil emulsion formed by emulsifying an aqueous phase with an oil phase in the presence of a surfactant comprising Span80, Tween80 and TritonX100.

57. The water-in-oil emulsion of claim 56 wherein said surfactant comprises 4.5% v/v Span80, 0.4% v/v Tween80 and 0.1% v/v TritonX100.