Method for in vitro molecular evolution of protein function

• US 6,495,321 B1
• Filed: 04/06/2000
• Issued: 12/17/2002
• Est. Priority Date: 06/16/1997
• Status: Expired due to Term

• Current Assignee: Alligator Bioscience AB
• Original Assignee: BioInvent International AB
• Inventors: Soderlind, Ulf Hans Eskil, Borrebaeck, Carl Arne Krister, Ottosson, Rebecka Ingrid Camilla
• Primary Examiner(s): Horlick, Kenneth R.
• Application Number:
  US09/445,649
• Time in Patent Office: 985 Days
• Field of Search: 435/6, 435/91.2, 435/68.1, 435/69.1, 435/91.1, 435/199, 435/810, 530/350, 536/23.1, 536/24.2, 536/24.3, 536/24.33, 514/44
• US Class Current: 435/6.16
• CPC Class Codes: C12N 15/1027 by DNA shuffling, e.g. RSR, STEP, RPR
• CPC Class Codes: C12N 15/1037 Screening libraries presented on the surface of microorganisms, e.g. phage display, E. coli display
• CPC Class Codes: C12Q 1/6811 Selection methods for production or design of target specific oligonucleotides or binding molecules
• CPC Class Codes: C12Q 1/6858 Allele-specific amplification
• CPC Class Codes: C12Q 2521/319 Exonuclease
• 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

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 polynucleofide sequence which encodes for a polypeptide having desired characteristics. This method may be usefully applied to the generation of new antibodies or parts thereof having modified characteristics as compared to the parent antibody.

Claims

1. A method for generating at least one polynucleotide sequence from a parent polynucleotide sequence encoding at least one protein motif, comprising the steps of

a) digesting the parent polynucleotide sequence with an exonuclease to generate a population of fragments;

b) contacting said fragments with an undigested template polynucleotide sequence under annealing conditions;

c) amplifying the fragments that anneal to the template in step b) to generate at least one polynucleotide sequence encoding at least one protein motif having altered characteristics as compared to the at least one protein motif encoded by said parent polynucleotide.

2. A method according to claim 1 wherein the parent polynucleotide is double-stranded and the method further comprises the step of generating single-stranded polynucleotide sequence from said double-stranded fragments prior to step b).

3. A method according to claim 1 wherein template polynucleotide sequence is the parent polynucleotide sequence.

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

11. A method for preparing a pharmaceutical composition which comprises, following the identification of a polynucleotide with desired characteristics by a method according to claim 1, adding said polynucleotide to a pharmaceutically acceptable carrier.

12. A method for preparing a pharmaceutical composition which comprises, following the identification of a polypeptide with desired characteristics by a method according to claim 8, adding said polypeptide to a pharmaceutically acceptable carrier.

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

4. A method for generating at least one polynucleotide sequence from a parent polynucleotide sequence encoding at least one protein motif, in which said polynucleotide sequence has altered sequence at its termini as compared to said parent polynucleotide sequence comprising the steps of

(a) digesting said parent polynucleotide sequence with an exonuclease to generate a population of fragments;

(b) contacting said fragments with an undigested template polynucleotide sequence which is a variant of said parent polynucleotide sequence, under annealing conditions;

(c) amplifying the fragments that anneal to the template in step b) to generate at least one polynucleotide sequence encoding at least one protein motif encoded by said parent polynucleotide.

6. A method according to claim 4 wherein the exonuclease is BAL31.

7. A method according to claim 4 wherein the parent polynucleotide sequence encodes an antibody or fragment thereof.

9. A method according to claim 4 further comprising the step of screening the at least one polynucleotide generated in step c) for desired characteristics.

10. A method according to claim 4 further comprising the step of expressing the at least one polynucleotide generated in step c) and screening the resulting polypeptide for desired characteristics.

14. A process which comprises, following the identification of a polypeptide by a method according to claim 9, the manufacture of that polypeptide, in whole or in part, optionally in conjunction with additional polypeptides.

15. A process according to claim 14 wherein the polypeptide is an antibody or fragment thereof.

16. A process according to claim 14 wherein the polypeptide is an enzyme.

5. A method for generating at least one polynucleotide sequence from a parent polynucleotide sequence encoding at least one protein motif, in which said polynucleotide sequence has altered sequence at its center as compared to said parent polynucleotide sequence comprising the steps of

(a) digesting a population of variant polynucleotide sequences with an exonuclease to generate a population of fragments;

(b) contacting said fragments with said parent polynucleotide sequence under annealing conditions, said parent polynucleotide sequence being undigested;

(c) amplifying the fragments that anneal to the parent in step b) to generate at least one polynucleotide sequence encoding at least one protein motif encoded by said parent polynucleotide.

17. A method according to claim 5 further comprising the step of screening the at least one polynucleotide generated in the step c) for desired characteristics.

18. A method according to claim 5 further comprising the step of expressing the at least one polynucleotide generated in step c) and screening the resulting polypeptide for desired characteristics.