DNA mutagenesis by random fragmentation and reassembly

• US 5,830,721 A
• Filed: 03/04/1996
• Issued: 11/03/1998
• Est. Priority Date: 02/17/1994
• Status: Expired due to Term

• Current Assignee: Codexis, Inc.
• Original Assignee: Affymax Incorporated
• Inventors: Stemmer, Willem P. C., Crameri, Andreas
• Primary Examiner(s): Jones, W. Gary
• Assistant Examiner(s): WHISENANT, ETHAN C
• Application Number:
  US08/537,874
• Time in Patent Office: 974 Days
• Field of Search: 435/6, 435/172.1, 435/91.2, 435/23.1, 536/24.3
• US Class Current: 506/10
• CPC Class Codes: C07K 14/43595 from coelenteratae, e.g. medusae
• CPC Class Codes: C07K 14/545 IL-1
• 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 2317/565 Complementarity determining region [CDR]
• CPC Class Codes: C07K 2317/622 Single chain antibody (scFv)
• CPC Class Codes: C12N 15/1027 by DNA shuffling, e.g. RSR, STEP, RPR
• CPC Class Codes: C12N 15/1034 Isolating an individual clone by screening libraries
• CPC Class Codes: C12N 15/1037 Screening libraries presented on the surface of microorganisms, e.g. phage display, E. coli display
• CPC Class Codes: C12N 9/2471 Beta-galactosidase (3.2.1.23), i.e. exo-(1-->4)-beta-D-galactanase
• CPC Class Codes: C12N 9/86 acting on amide bonds in cyclic amides, e.g. penicillinase (3.5.2)
• CPC Class Codes: C12Q 1/68 involving nucleic acids
• CPC Class Codes: C12Q 1/6811 Selection methods for production or design of target specific oligonucleotides or binding molecules
• CPC Class Codes: C12Y 302/01023 Beta-galactosidase (3.2.1.23), i.e. exo-(1-->4)-beta-D-galactanase
• CPC Class Codes: C12Y 305/02006 Beta-lactamase (3.5.2.6)
• 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
• CPC Class Codes: C40B 40/08 Libraries containing RNA or DNA which encodes proteins, e.g. gene libraries
• CPC Class Codes: C40B 50/06 Biochemical methods, e.g. using enzymes or whole viable microorganisms

Abstract

A method for generating libraries of displayed peptides and/or antibodies (Abs) suitable for affinity interaction screening or phenotypic screening comprising: (i) obtaining selected library members comprising a displayed peptide and/or Ab and the corressponding polynucleotide (PN), or copies of it, (ii) pooling and fragmenting the PN, or copies of it, to form fragments, (iii) performing PCR amplification and thereby homologously recombining the fragments to form a shuffled pool of recombined PNs, which are not present in the selected library of (i).

Claims

1. A method for forming a mutagenized double-stranded polynucleotide from a template double-stranded polynucleotide, comprising:

a) providing a population of double-stranded overlapping fragments of the template double-stranded polynucleotide and one or more single or double-stranded oligonucleotides, wherein said oligonucleotides comprise an area of identity and an area of heterology to the template double-stranded polynucleotide;

b) denaturing the resultant mixture of double-stranded overlapping fragments and oligonucleotides into single-stranded fragments;

c) incubating the resultant population of single-stranded fragments with a polymerase under conditions which result in the annealing of said single-stranded fragments at said areas of identity to form pairs of annealed fragments, said areas of identity being sufficient for one member of a pair to prime replication of the other thereby forming mutagenized double-stranded polynucleotides; and

d) repeating steps (b) and (c) for at least two further cycles, wherein the resultant mixture in step (b) of a further cycle includes the mutagenized double-stranded polynucleotides from step (c) of the previous cycle, and the further cycle forms further mutagenized double-stranded polynucleotides whereby the average length of the mutagenized polynucleotides increases in each cycle.

2. The method of claim 1 wherein the concentration of a single species of double-stranded overlapping fragment in the population of double-stranded overlapping fragments is less than 1% by weight of the total DNA.

3. The method of claim 1 wherein the number of different specific overlapping double-stranded fragments comprises at least about 100.

4. The method of claim 1 wherein the size of the double-stranded overlapping fragments is from about 5 bp to 5 kb.

5. The method of claim 1 wherein the size of the template double-stranded polynucleotide comprises from 50 bp to 100 kb.

6. The method of claim 1, wherein the double-stranded template polynucleotide encodes a wild-type protein;

and the method further comprises

f) expressing a mutant form of the wildtype protein from a further mutagenized double-stranded polynucleotide.

7. The method of claim 6 further comprising selecting the desired recombinant protein from a population of recombinant proteins encoded by further mutagenized polynucleotides produced by repeating cycles of (b) and (c).

28. The method of claim 1, wherein step d) comprises repeating steps b)-c) for at least 10 cycles.

8. A method for obtaining a chimeric polynucleotide sequence comprising:

a) treating a sample comprising different double-stranded template polynucleotides wherein said different template polynucleotides contain areas of identity and areas of heterology under conditions whereby overlapping double-stranded fragments of a desired size of said different double-stranded template polynucleotides are formed;

b) denaturing the resultant overlapping double-stranded fragments of said different double-stranded template polynucleotides contained in the treated sample produced by step (a) into single-stranded fragments;

c) incubating the resultant single-stranded fragments with polymerase under conditions which provide for the annealing of the single-stranded fragments at the areas of identity to form pairs of annealed fragments, said areas of identity being sufficient for one member of a pair to primer replication of the other thereby forming chimeric double-stranded polynucleotide sequences comprising template polynucleotide sequences; and

d) repeating steps (b) and (c) for at least two cycles, wherein the resultant mixture in step (b) of a cycle includes the chimeric double-stranded polynucleotide sequences in step (c), and the further cycle forms a further chimeric polynucleotide sequences whereby and the average length of chimeric polynucleotide sequences increases in each cycle.

9. The method of claim 8 wherein the concentration of a specific double-stranded overlapping fragment in the double-stranded overlapping fragments of a desired size in step (a) is less than 1% by weight of the total DNA.

10. The method of claim 8 where the number of different double-stranded overlapping fragments of a desired size in step (a) comprises at least about 100.

11. The method of claim 8 wherein the size of the double-stranded overlapping fragments of a desired size is from about 5 bp to 5 kb.

12. The method of claim 8 wherein the size of the different template double-stranded polynucleotides is from 50 bp to 100 kb.

13. A method for generating libraries of displayed peptides or displayed antibodies suitable for affinity interaction screening or phenotypic screening, the method comprising:

(1) obtaining a first plurality of selected library members comprising a displayed peptide or displayed antibody and an associated polynucleotide encoding said displayed peptide or displayed antibody, and obtaining said associated polynucleotides or copies thereof wherein said associated polynucleotides comprise a region of substantially identical sequence, and

(2) pooling and fragmenting said associated polynucleotides or copies to form a population of double-stranded fragments thereof

3) denaturing the population of double-stranded fragments into single-stranded fragments;

c) incubating the resultant population of single-stranded fragments with a polymerase under conditions which result in the annealing of said single-stranded fragments at areas of overlap to form pairs of annealed fragments, whereby one member of a pair primes replication of the other thereby forming recombinant polynucleotides;

d) repeating steps b) and c) for at least two further cycles wherein the resultant mixture in step (b) of a further cycle includes the recombinant polynucleotides from step c) of the previous cycle, whereby the average length of polynucleotides increases in each cycle to form a shuffled pool of recombined polynucleotides, whereby a substantial fraction of the recombined polynucleotides of said shuffled pool are not present in the associated polynucleotides in step (1) and the shuffled pool of recombined polynucleotides encodes the libraries of displayed peptides or displayed antibodies suitable for affinity interaction screening or phenotypic screening.

14. The method of claim 13, further comprising introducing mutations into said associated polynucleotides or copies.

15. The method of claim 14, wherein the mutations are introduced by performing PCR amplification.

16. The method of claim 15, wherein the PCR amplification is error-prone PCR.

17. The method of claim 13, comprising the additional step of screening the library members of the shuffled pool to identify individual shuffled library members having the ability to bind with a predetermined macromolecule.

18. The method of claim 13, wherein the first plurality of selected library members is obtained by selecting for a phenotypic characteristic other than binding affinity for a predetermined molecule.

19. The method of claim 13, wherein the first plurality of selected library members is pooled and fragmented and homologously recombined by PCR in vitro.

20. The method of claim 13, wherein the first plurality of selected library members is pooled and fragmented in vitro, the resultant fragments transferred into a host cell or organism and homologously recombined to form shuffled library members in vivo.

21. The method of claim 13, wherein the first plurality of selected library members is cloned or amplified on episomally replicable vectors, a multiplicity of said vectors is transferred into a cell and homologously recombined to form shuffled library members in vivo.

25. The method of claim 13 for generating libraries of displayed antibodies, wherein the shuffled pool of recombined polynucleotides comprises novel combinations of CDRs, whereby a substantial fraction of the recombined polynucleotides of said shuffled pool comprise CDR combinations are not present in the first plurality of selected library members.

26. The method of claim 25, comprising the additional step wherein the shuffled pool is subjected to affinity screening to select shuffled library members which bind to a predetermined epitope and thereby selecting a plurality of selected shuffled library members.

27. The method of claim 26, comprising the additional step of shuffling the plurality of selected shuffled library members and screening, from 1 to about 1000 cycles.

22. A method for generating libraries of displayed peptides or displayed antibodies suitable for affinity interaction screening or phenotypic screening, the method comprising:

(1) obtaining a first plurality of selected library members comprising a displayed peptide or displayed antibody and an associated polynucleotide encoding said displayed peptide or displayed antibody, and obtaining said associated polynucleotides or copies thereof wherein said associated polynucleotides comprise a region of substantially identical sequence, and

(2) cloning or amplifying said associated polynucleotides or copies on episomally replicable vectors and transferring a multiplicity of said vectors into a cell and homologously recombining to form shuffled library members in vivo encoding the libraries of displayed peptide or displayed antibodies suitable for affinity interaction screening or phenotypic screening.

23. The method of claim 22, further comprising introducing mutations into said associated polynucleotides or copies thereof.

24. The method of claim 22, wherein said episomally replicable vectors comprise a direct repeat of a plurality of associated polynucleotides or copies thereof.