Method for in vitro molecular evolution of protein function

• US 6,989,250 B2
• Filed: 04/08/2002
• Issued: 01/24/2006
• Est. Priority Date: 01/24/1997
• Status: Expired due to Term

• Current Assignee: BioInvent International AB
• Original Assignee: BioInvent International AB
• Inventors: Soderlind, Ulf Hans Eskil, Borrebaeck, Carl Arne Krister
• Primary Examiner(s): LU, FRANK WEI MIN
• Application Number:
  US10/118,100
• Publication Number:
  US 20030077613A1
• Time in Patent Office: 1,387 Days
• Field of Search: 435/6, 435/91.1, 435/91.2, 435/183, 435/252.3, 435/320.1, 435/455, 435/463, 435/465, 436/94, 536/23.1, 536/24.3, 536/24.33, 536/25.3, 536/25.32
• US Class Current: 435/91.2
• CPC Class Codes: C07K 16/00 Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies antibodies with enzymatic activity, e.g. abzymes C12N9/0002
• CPC Class Codes: C07K 16/44 against material not provided for elsewhere , e.g. haptens, metals, DNA, RNA, amino acids
• CPC Class Codes: C07K 2317/622 Single chain antibody (scFv)
• CPC Class Codes: C07K 2317/92 Affinity (KD), association rate (Ka), dissociation rate (Kd) or EC50 value
• CPC Class Codes: C12N 15/102 Mutagenizing nucleic acids
• CPC Class Codes: C12N 15/1027 by DNA shuffling, e.g. RSR, STEP, RPR
• CPC Class Codes: C12N 15/1031 mutagenesis by gene assembly, e.g. assembly by oligonucleotide extension PCR
• 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 15/1086 Preparation or screening of expression libraries, e.g. reporter assays
• CPC Class Codes: Y10T 436/143333 Saccharide [e.g., DNA, etc.]

Abstract

The present invention relates to a method for in vitro creation of molecular libraries evolution of protein function. Particularly, it relates to variability and modification of protein function by shuffling polynucleotide sequence segments. A protein of desired characteristics can be obtained by incorporating variant peptide regions (variant motifs) into defined peptide regions (scaffold sequence). The variant motifs can be obtained from parent DNA which has been subjected to mutagenesis to create a plurality of differently mutated derivatives thereof or they can be obtained from in vivo sequences. These variant motifs can then be incorporated into a scaffold sequence and the resulting coded protein screened for desired characteristics. This method is ideally used for obtaining antibodies with desired characteristics by isolating individual CDR DNA sequences and incorporating them into a scaffold which may, for example, be from a totally different antibody.

Claims

1. A method of generating an assembled polynucleotide sequence encoding a protein of desired characteristics comprising the steps of:

a) providing at least one polynucleotide sequence comprising one or more variant polynucleotide sequences encoding one or more variant protein motifs;

b) providing one or more pairs of defined oligonucleotides, each pair representing spaced apart locations on the at least one polynucleotide sequence of step a), each pair binding adjacent to a variant polynucleotide sequence encoding a variant protein motif;

c) using the pairs of defined oligonucleotides as amplification primers for PCR to amplify the variant polynucleotide sequences encoding the variant protein motifs of the at least one polynucleotide sequence of step a) and performing PCR amplification on the at least one polynucleotide sequences of step a);

d) obtaining one or more single-stranded polynucleotide sequences from the amplified polynucleotide sequences in step c);

e) providing one or more unmutated, specifically selected, scaffold polynucleotide sequences encoding one or more unmutated peptide regions; and

f) annealing said one or more single-stranded polynucleotide sequences from step d) with the unmutated, specifically selected, scaffold polynucleotide sequences from step e) such that annealed polynucleotides with one or more gaps is formed, and filling the one or more gaps present in the annealed polynucleotides, thereby generating one or more assembled polynucleotide sequences.

2. The method according to claim 1 wherein providing the polynucleotide sequence in step (a) comprises subjecting a polynucleotide sequence encoding one or more variant protein motifs to mutagenesis to create a plurality of differently mutated derivatives thereof.

3. The method according to claim 2, wherein the at least one polynucleotide is subjected to error-prone PCR.

4. The method according to claim 3, further comprising the step of expressing a protein encoded by the assembled polynucleotide sequence and screening for desired properties.

5. The method according to claim 4, wherein the defined oligonucleotides are single stranded.

6. The method according to claim 4, wherein one of said pair of defined oligonucleotides is linked to a member of a specific binding pair (MSBP).

7. The method according to claim 6, further comprising the steps of isolating the amplified polynucleotide sequence by binding the MSBP of the amplified polynucleotide sequence to its specific binding partner.

8. The method according to claim 6, wherein the MSBP is biotin.

9. The method according to claim 6, wherein the specific binding partner is streptavidin.

10. The method according to claim 4, wherein the at least one polynucleotide sequence of step a) encodes an antibody or antibody fragment capable of binding an antigen or other binding partner of the antibody or the antibody fragment.

11. The method according to claim 3, wherein the defined oligonucleotides are single stranded.

12. The method according to claim 11, wherein one of said pair of defined oligonucleotides is linked to a member of a specific binding pair (MSBP).

13. The method according to claim 11, wherein the at least one polynucleotide sequence of step a) encodes an antibody or antibody fragment capable of binding an antigen or other binding partner of the antibody or the antibody fragment.

14. The method according to claim 3, wherein one of said pair of defined oligonucleotides is linked to a member of a specific binding pair (MSBP).

15. The method according to claim 14, further comprising the steps of isolating the amplified polynucleotide sequence by binding the MSBP of the amplified polynucleotide sequence to its specific binding partner.

16. The method according to claim 15, wherein the at least one polynucleotide sequence of step a) encodes an antibody or antibody fragment capable of binding an antigen or other binding partner of the antibody or the antibody fragment.

17. The method according to claim 14, wherein the MSBP is biotin.

18. The method according to claim 14, wherein the MSBP is streptavidin.

19. The method according to claim 14, wherein the at least one polynucleotide sequence of step a) encodes an antibody or antibody fragment capable of binding an antigen or other binding partner of the antibody or the antibody fragment.

20. The method according to claim 3 wherein the at least one polynucleotide sequence of step a) encodes an antibody or antibody fragment capable of binding an antigen or other binding partner of the antibody or the antibody fragment.

21. The method according to claim 1, further comprising the step of expressing a protein encoded by the assembled polynucleotide sequence and screening for desired properties.

22. The method according to claim 1, wherein the defined oligonucleotides are single-stranded.

23. The method according to claim 1, wherein one of said pair of defined oligonucleotides is linked to a member of a specific binding pair (MSBP).

24. The method according to claim 23, further comprising the steps of isolating the amplified polynucleotide sequence by binding the MSBP of the amplified polynucleotide sequence to its specific binding partner.

25. The method according to claim 24, wherein the MSBP is biotin.

26. The method according to claim 24, wherein the specific binding partner is streptavidin.

27. The method according to claim 1, wherein the at least one polynucleotide sequence of step a) encodes an antibody or antibody fragment capable of binding an antigen or other binding partner of the antibody or the antibody fragment.

28. The method according to claim 1, comprising obtaining a plurality of polynucleotides each encoding one or more variant protein motifs.

29. A method of creating a polynucleotide library comprising the steps of:

a) providing at least one polynucleotide sequence comprising one or more variant polynucleotide sequences encoding one or more variant protein motifs;

b) providing one or more pairs of defined oligonucleotides, each pair representing spaced apart locations on the at least one polynucleotide sequence of step a), each pair binding adjacent to a variant polynucleotide sequence encoding a variant protein motif;

c) using the pairs of defined oligonucleotides as amplification primers for PCR to amplify the variant polynucleotide sequences encoding the variant protein motifs of the at least one polynucleotide sequence of step a) and performing PCR amplification on the at least one polynucleotide sequences of step a);

d) obtaining one or more single-stranded polynucleotide sequences from the amplified polynucleotide sequences in step c);

e) providing one or more unmutated, specifically selected, scaffold polynucleotide sequences encoding one or more unmutated scaffold peptide regions;

f) annealing said one or more single-stranded polynucleotide sequences from step d) with the unmutated, specifically selected, scaffold polynucleotide sequences from step e) such that annealed polynucleotides with one or more gaps is formed, and filling the one or more gaps present in the annealed polynucleotides, thereby generating one or more assembled polynucleotide sequences; and

g) inserting said assembled polynucleotide sequences into suitable vectors, thereby creating said polynucleotide library.

30. The method according to claim 29 wherein providing the polynucleotide sequence in step (a) comprises subjecting a polynucleotide encoding one or more variant protein motifs to mutagenesis to create a plurality of differently mutated derivatives thereof.

31. The method according to claim 30, wherein the at least one polynucleotide sequence is subjected to error prone PCR.

32. The method according to claim 31, further comprising the steps of selecting and expressing said polynucleotide sequences to obtain a library of protein sequences.

33. The method according to claim 32, further comprising screening the library of protein sequences for a protein of desired characteristics.

34. The method according to claim 33, wherein one of said pair of defined oligonucleotides is linked to a member of a specific binding pair (MSBP).

35. The method according to claim 34, further comprising the steps of isolating the amplified polynucleotide sequence by binding the MSBP of the amplified polynucleotide sequence to its specific binding partner.

36. The method according to claim 32, wherein one of said pair of oligonucleotides is linked to a member of a specific binding pair (MSBP).

37. The method according to claim 36, further comprising the steps of isolating the amplified polynucleotide sequence by binding the MSBP of the amplified polynucleotide sequence to its specific binding partner.

38. The method according to claim 31, further comprising selecting and expressing said polynucleotide sequences to obtain proteins of desired characteristics.

39. The method according to claim 31, wherein one of said pair of oligonucleotides is linked to a member of a specific binding pair (MSBP).

40. The method according to claim 39, further comprising the steps of isolating the amplified polynucleotide sequence by binding the MSBP of the amplified polynucleotide sequence to its specific binding partner.

41. The method according to claim 29, further comprising selecting and expressing said polynucleotide sequences to obtain proteins of desired characteristics.

42. The method according to claim 41, wherein one of said pair of defined oligonucleotides is linked to a member of a specific binding pair (MSBP).

43. The method according to claim 42, further comprising the steps of isolating the amplified polynucleotide sequence by binding the MSBP of the amplified polynucleotide sequence to its specific binding partner.

44. The method according to claim 29, wherein one of said pair of defined oligonucleotides is linked to a member of a specific binding pair (MSBP).

45. The method according to claim 44, further comprising the steps of isolating the amplified polynucleotide sequence by binding the MSBP of the amplified polynucleotide sequence to its specific binding partner.

46. The method according to claim 29, further comprising the steps of selecting and expressing said polynucleotide sequences to obtain a library of protein sequences.

47. The method according to claim 46, further comprising screening the library of protein sequences for a protein of desired characteristics.

48. The method according to claim 29, comprising obtaining a plurality of polynucleotides each encoding one or more variant protein motifs.

49. A method of creating a polynucleotide library comprising the following steps:

a) providing a at least one polynucleotide encoding one or more variant protein motifs;

b) providing one or more pairs of defined oligonucleotides, each pair representing spaced apart locations on the at least one polynucleotide sequence of step a), each pair binding adjacent to a variant polynucleotide sequence encoding a variant protein motif;

c) using the pairs of defined oligonucleotides as amplification primers for PCR to amplify the variant polynucleotide sequences encoding the variant protein motifs of the at least one polynucleotide sequence of step a) and performing PCR amplification on the at least one polynucleotide sequence of step a);

d) obtaining single-stranded polynucleotide sequences from the amplified polynucleotide sequences produced in step c);

e) providing one or more unmutated, specifically selected, scaffold polynucleotide sequences encoding one or more unmutated scaffold peptide regions; and

f) annealing one or more single-stranded polynucleotide sequences produced in step (d) with the unmutated, specifically selected, scaffold polynucleotide sequences from step (e) such that annealed polynucleotides with one or more gaps are formed, and filling the one or more gaps present in the annealed polynucleotides to assemble a library of polynucleotide sequences each encoding a protein comprising one or more variant protein motifs and one or more unmutated scaffold peptide regions.