Method for in vitro molecular evolution of protein function

• US 20060166198A1
• Filed: 05/16/2003
• Published: 07/27/2006
• Est. Priority Date: 05/17/2002
• Status: Active Grant

• Current Assignee: Alligator Bioscience AB
• Original Assignee: Alligator Bioscience AB
• Inventors: Borrebaeck, Carl Arne Krister, Carlsson, Roland, Furebring, Christina, Malmborg Hager, Ann-Christin
• Granted Patent: US 7,262,012 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
• 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: C12P 19/04 Polysaccharides, i.e. compounds containing more than five saccharide radicals attached to each other by glycosidic bonds

Abstract

A method for in vitro molecular evolution of protein function The invention provides a method for generating a polynucleotide sequence or population of sequences from parent single-stranded polynucleotide sequences encoding one or more protein motifs, comprising the steps of (a) providing a first population of single-stranded polynucleotide molecules and a second population of single-stranded polynucleotide molecules, the first and second populations together constituting plus and minus strands of parent polynucleotide sequences, (b) carrying out a reaction for digesting the first and second populations of single-stranded polynucleotide molecules with an exonuclease to generate corresponding populations of single-stranded polynucleotide fragments, (c) contacting said fragments generated from the plus strands with fragments generated from the minus strands and optionally, adding primer sequences that anneal to the 3′ and 5′ends of at least one of the parent polynucleotides under annealing conditions, and (d) amplifying the fragments that anneal to each other 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 polynucleotides, wherein, in step (b), 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. Preferably, 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, amount of single-stranded polynucleotide molecules and buffer composition of the reaction mixture.

Claims

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

a) providing a first population of single-stranded polynucleotide molecules and a second population of single-stranded polynucleotide molecules, the first and second populations together constituting plus and minus strands of parent polynucleotide sequences;

b) carrying out a reaction for digesting the first and second populations of single-stranded polynucleotide molecules with an exonuclease to generate corresponding populations of single-stranded polynucleotide fragments;

c) contacting said polynucleotide fragments generated from the plus strands with fragments generated from the minus strands; and

d) amplifying the fragments that anneal to each other 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 polynucleotides.

wherein, in step (b), 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 exonuclease used for digestion of the first population of single-stranded polynucleotide molecules is different from the exonuclease used for digestion of the second population of single-stranded polynucleotide molecules.

4. A method according to claim 3 wherein the exonuclease used for digestion of the first population of single-stranded polynucleotide molecules is a 3′

exonuclease and the exonuclease used for digestion of the second population of single-stranded polynucleotide molecules is a 5′

exonuclease.

5. 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.

6. 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.

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

8. 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.

9. 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.

10. 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.

11. A method according to claim 1 wherein the buffer composition of the reaction mixture used for digestion of the first population of single-stranded polynucleotide molecules is different from the buffer composition of the reaction mixture used for digestion of the second population of single-stranded polynucleotide molecules.

12. A method according to claim 1 wherein the amount of single-stranded polynucleotide molecules in the first population of single-stranded polynucleotide molecules is different from the amount of single-stranded polynucleotide molecules in the second population of single-stranded polynucleotide molecules.

13. A method according to claim 1 wherein the first population of single-stranded polynucleotide molecules constitutes the plus strands of parent polynucleotide sequences and the second population of single-stranded polynucleotide molecules constitutes the minus strands of parent polynucleotide sequences.

14. A method according to claim 1 wherein the polynucleotide molecules of step (a) are DNA molecules.

15. A method according to claim 1 wherein step c) further comprises adding primer sequences that anneal to the 3′

and/or 5′

ends of at least one of the parent polynucleotides under annealing conditions.

16. A method according to claim 1 wherein the exonuclease used to digest the first and/or second population of single-stranded polynucleotide molecules is selected from the group consisting of BAL 31, exonuclease I, exonuclease V, exonuclease VII, exonuclease T7 gene 6, bacteriophage lambda exonuclease and exonuclease Rec J_f.

17. A method according to claim 1 wherein a parent polynucleotide sequence or sequences has been subjected to mutagenesis.

18. A method according to claim 1 wherein one or both of the populations of fragments generated in step b) are subjected to mutagenesis.

19. A method according to claim 1 wherein the parent population or population of fragments has been subjected to mutagenesis is error prone PCR.

20. A method according to claim 1 wherein step b) is carried out to generate populations of single-stranded fragments of varying lengths.

21. A method according to claim 20 wherein step b) is controlled to generate a population of single-stranded fragments having an average length of more than approximately 50 nucleotides.

22. A method according to claim 1 further comprising the step of expressing at least one polynucleotide sequence generated in step d) to produce the encoded polypeptide.

23. A method according to claim 22 further comprising the step of testing the encoded polypeptide for desired characteristics.

24. A method according to claim 1 wherein the parent polynucleotide sequence encodes an antibody or fragment thereof.

25. A method according to claim 1 wherein the parent polynucleotide sequence encodes an enzyme.

26. A method according to claim 1 wherein the parent polynucleotide sequence encodes an antigen.

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

(a) generating variant forms of a parent polynucleotide using a method according to 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.

28. A polypeptide obtained by a method according to claim 27.

29. A pharmaceutical composition comprising a polypeptide according to claim 28 and a pharmaceutically acceptable carrier.

30. The polypeptide of claim 28 further comprising a pharmaceutically acceptable carrier.

31. Use of a polypeptide according to claim 28 in the preparation of a medicament for the treatment, therapy and/or diagnosis of a disease.

32. A process for preparing a pharmaceutical composition which comprises, following the identification of a polynucleotide and/or encoded polypeptide with desired characteristics by a method according to claim 1, adding said polynucleotide and/or encoded polypeptide to a pharmaceutically acceptable carrier.

33. A process which comprises, following identification of a polynucleotide and/or encoded polypeptide with desired characteristics by a method according to any one of claims 1 to 26, use of that polynucleotide and/or encoded polypeptide, in whole or in part, in medicine.

34. A process as claimed in claim 33 wherein the use in medicine is in the treatment, therapy and/or diagnosis of a disease.

35. A process which comprises, following identification of a polynucleotide with desired characteristics according to the method of claim 1, the use of that polynucleotide in the detection and/or amplification of a target polynucleotide in a sample.