Methods for generating polynucleotides having desired characteristics by iterative selection and recombination

• US 7,868,138 B2
• Filed: 02/01/2008
• Issued: 01/11/2011
• Est. Priority Date: 02/17/1994
• Status: Expired due to Fees

• Current Assignee: Codexis, Inc.
• Original Assignee: Codexis, Inc.
• Inventors: Crameri, Andreas, Stemmer, Willem P. C.
• Primary Examiner(s): Desai; Anand U
• Assistant Examiner(s): LIU, SAMUEL W
• Application Number:
  US12/012,528
• Publication Number:
  US 20100286369A1
• Time in Patent Office: 1,075 Days
• Field of Search: None
• US Class Current: 530/350
• 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 15/1037 Screening libraries presented on the surface of microorganisms, e.g. phage display, E. coli display
• 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 15/52 Genes encoding for enzymes or proenzymes
• CPC Class Codes: C12N 15/64 General methods for preparing the vector, for introducing it into the cell or for selecting the vector-containing host
• 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: 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: Y10S 435/876 Pseudomonas fluorescens

Abstract

A method for DNA reassembly after random fragmentation, and its application to mutagenesis of nucleic acid sequences by in vitro or in vivo recombination is described. In particular, a method for the production of nucleic acid fragments or polynucleotides encoding mutant proteins is described. The present invention also relates to a method of repeated cycles of mutagenesis, shuffling and selection which allow for the directed molecular evolution in vitro or in vivo of proteins.

Claims

1. A mutant green fluorescent protein (GFP) with enhanced fluorescent intensity as compared to wild type Aequorea victoria GFP, said mutant protein comprising point mutations at two or more residues corresponding to residues selected from the group consisting of F99, M153, and E172 in wild type A. victoria GFP, wherein said mutations comprise replacement of hydrophobic residues with more hydrophilic residues.

2. The mutant protein of claim 1, wherein said mutations comprise mutations at residues corresponding to F99, M153, and E172 in wild type A. victoria GFP.

3. The mutant protein of claim 2, wherein said mutations comprise mutation of the residue corresponding to M153 in wild type A. victoria GFP to threonine.

4. The mutant protein of claim 2, wherein said mutations comprise mutation of the residue corresponding to E172 in wild type A. victoria GFP to glycine.

5. The mutant protein of claim 2, wherein said mutations comprise mutation of the residue corresponding to F99 in wild type A. victoria GFP to serine;

mutation of the residue corresponding to M153 in wild type A. victoria GFP to threonine; and

mutation of the residue corresponding to E172 in wild type A. victoria GFP to glycine.

6. The mutant protein of claim 2, wherein said mutations comprise mutation of the residue corresponding to F99 in wild type A. victoria GFP to serine.