Method for in vitro molecular evolution of protein function

• US 20030148353A1
• Filed: 12/17/2002
• Published: 08/07/2003
• Est. Priority Date: 06/16/1998
• Status: Active Grant

• Current Assignee: Alligator Bioscience AB
• Original Assignee: BioInvent International AB
• Inventors: Soderlind, Ulf Hans Eskil, Borrebaeck, Carl Arne Krister, Furebring, Christina, Ottosson, Rebecka Ingrid Camilla, Malmborg-Hager, Ann-Christin
• Granted Patent: US 7,153,655 B2
• Time in Patent Office: Days
• US Class Current: 435/6
• CPC Class Codes: C12N 15/1027 by DNA shuffling, e.g. RSR, STEP, RPR

Abstract

The present invention relates to a method for in vitro evolution of protein function. In particular, the method relates to the shuffling of nucleotide segments obtained from exonuclease digestion. The present inventors have shown that polynucleotide fragments derived from a parent polynucleotide sequence digested with an exonuclease can be combined to generate a polynucleotide sequence which encodes for a polypeptide having desired characteristics. This method may be usefully applied to the generation of new proteins (e.g., antibodies and enzymes) or parts thereof having modified characteristics as compared to the parent protein.

Claims

1. A method for generating a polynucleotide sequence or population of sequences from at least one parent polynucleotide sequence encoding one or more protein motifs, comprising the steps of

a) digesting the at least one parent polynucleotide sequence with at least one exonuclease to generate at least one population of fragments, wherein said digesting with at least one exonuclease comprises digesting a first parent polynucleotide sequence with a first exonuclease to produce a first population of fragments and digesting a second parent polynucleotide sequence with a second exonuclease to produce a second population of fragments;

b) contacting said first and second population of fragments, wherein said fragments of said first and second population of fragments anneal;

c) amplifying the annealed fragments of step b) to generate at least one polynucleotide sequence encoding one or more protein motifs having altered characteristics as compared to the one or more protein motifs encoded by said parent polynucleotide

wherein in step (a) at least one parameter of the reaction used for digestion of the first population of single stranded polynucleotide molecules is different from the equivalent parameter(s) used in the reaction for digestion of the second population of single stranded polynucleotide molecules.

2. A method according to claim 1 wherein the reaction parameter is selected from exonuclease type, exonuclease concentration, reaction volume, duration of the digestion reaction, temperature of the reaction mixture, pH of the reaction mixture, length of parent single stranded polynucleotide sequences, the amount of single stranded polynucleotide molecules and the buffer composition of the reaction mixture.

3. A method according to claim 1, wherein the at least one parent polynucleotide sequence is double-stranded and the method further comprises the step of generating single-stranded plus and minus strand polynucleotide sequence from said double-stranded fragments prior to step a).

4. A method according to claim 3, wherein said single-stranded plus and minus strand polynucleotide sequences are isolated to generate a plus strand population and a minus strand population prior to step a).

5. A method according to claim 1, wherein the at least one parent polynucleotide sequence is single-stranded.

6. A method according to claim 1, wherein the first parent polynucleotide is at least one single-stranded plus polynucleotide sequence and the second parent polynucleotide is at least one single-stranded minus polynucleotide sequence.

7. A method according to claim 3 wherein the single-stranded plus polynucleotide sequence is digested with a first exonuclease to produce a first population of single-stranded plus fragments and the single-stranded minus polynucleotide sequence is digested with a second exonuclease to produce a second population of single-stranded minus fragments.

8. A method according to claim 1 wherein the first and second exonuclease are the same.

9. A method according to claim 1 wherein the first and the second exonuclease are different.

10. A method according to claim 1, wherein the at least one parent polynucleotide sequence has been subjected to mutagenesis.

11. A method according to claim 1, wherein the population of fragments generated in step b) is subjected to mutagenesis.

12. A method according to claim 10, wherein the mutagenesis is error prone mutagenesis.

13. A method according to claim 1, wherein said first and second exonuclease are selected from the group consisting BAL31, Exonuclease I, Exonuclease V, Exonuclease VII, T7 gene 6, and RecJ exonuclease.

14. A method according to claim 1, wherein said first parent polynucleotide sequence is digested with said first exonuclease for a first incubation time to produce a first population of fragments and said second parent polynucleotide sequence is digested with said second exonuclease for a second incubation time to produce a second population of fragments.

15. A method according to claim 14, wherein said first and second incubation time are the same.

16. A method according to claim 13, wherein said first and second incubation time are different.

17. A method according to claim 1, further comprising digesting a third parent polynucleotide sequence with a third exonuclease to produce a third population of fragments and contacting said third population of fragments to said first and second population of fragments in step b), wherein said fragments of said first, second, and third population of fragments anneal.

18. A method according to claim 1 wherein at least one parent polynucleotide sequence encodes an antibody or fragment thereof.

19. A method according to claim 1 wherein at least one parent polynucleotide sequence encodes an enzyme.

20. A method according to claim 1 further comprising the step of screening the at least one polynucleotide generated in step c) for desired characteristics.

21. A method according to claim 1 further comprising the step of expressing the at least one polynucleotide generated in step c) and screening the resulting polypeptide for desired characteristics.

22. A method according to claim 1, wherein the exonuclease concentration used for digestion of the first population of single stranded polynucleotide molecules is different from the exonuclease concentration used for digestion of the second population of single stranded polynucleotide molecules.

23. A method according to claim 1, wherein the reaction volume used for digestion of the first population of single stranded polynucleotide molecules is different from the reaction volume used for digestion of the second population of single stranded polynucleotide molecules.

24. A method according to claim 1, wherein the temperature of the reaction mixture used for digestion of the first population of single stranded polynucleotide molecules is different from the temperature of the reaction mixture used for digestion of the second population of single stranded polynucleotide molecules.

25. A method according to claim 1, wherein the pH of the reaction mixture used for digestion of the first population of single stranded polynucleotide molecules is different from the pH of the reaction mixture used for digestion of the second population of single stranded polynucleotide molecules.

26. A method according to claim 1, wherein the length of the polynucleotides in the first population of single stranded polynucleotide molecules is different from the length of the polynucleotides in the second population of single stranded polynucleotide molecules.

27. A method for preparing a pharmaceutical composition which comprises, identifying of a polynucleotide with desired characteristics as claimed in claim 1 and adding said polynucleotide to a pharmaceutically acceptable carrier.

28. The method of claim 27, wherein said polynucleotide is translated into a polypeptide having desired characteristics and said polypeptide is added to a pharmaceutically acceptable carrier.

29. A process which comprises, following the identification of a polynucleotide by the method of claim 1, the manufacture of that polynucleotide, in whole or in part, optionally in conjunction with an additional polynucleotide sequence.

30. The method according to claim 28 wherein the polypeptide is an antibody or fragment thereof.

31. The method according to claim 28 wherein the polypeptide is an enzyme.

32. A method of detecting and/or amplifying a target polynucleotide in a sample comprising contacting said sample with a polynucleotide having been identified by a method according to claim 1, said identified polynucleotide optionally being conjugated with an additional polynucleotide sequence.

33. A method as claimed in claim 1, wherein a plurality of populations of single stranded fragments of varying lengths are generated following digestion in step a).

34. A method according to claim 33, wherein said digestion is controlled to generate a population of single-stranded fragments having an average length of of more than approximately 50 nucleotides.

38. A method for making a polypeptide having desired properties, the method comprising the following steps:

(a) generating variant forms of a parent polynucleotide as claimed in claim 1;

(b) expressing the variant polynucleotides produced in step (a) to produce variant polypeptides;

(c) screening the variant polypeptides for desired properties; and

(d) selecting a polypeptide having desired properties from the variant polypeptides.

39. A polypeptide obtained by a method according to claim 38.

35. A novel green fluorescent protein comprising the amino acid sequence consisting of SEQ ID NO:

15.

36. A nucleic acid sequence encoding SEQ ID NO:

15.

37. A nucleic acid sequence comprising SEQ ID NO:

14.