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

• US 6,602,986 B1
• Filed: 11/15/2000
• Issued: 08/05/2003
• Est. Priority Date: 02/17/1994
• Status: Expired due to Fees

• Current Assignee: Codexis, Inc.
• Original Assignee: Maxygen Inc.
• Inventors: Crameri, Andreas, Stemmer, Willem P. C.
• Primary Examiner(s): PARK, HANKYEL
• Application Number:
  US09/713,920
• Time in Patent Office: 993 Days
• Field of Search: 530/387.3, 530/388.22
• US Class Current: 530/387.3
• 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 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. An antibody comprising a heavy chain comprising three nonnaturally occuring CDR regions, and a light chain comprising three nonnaturally occurring CDR regions, wherein the antibody specifically binds to a human antigen.

3. The antibody of claim 1 that binds to a human antigen selected from the group consisting of CD4, CD8, IL-2 receptor, EGF receptor and PDGF receptor.

2. An antibody comprising a heavy chain comprising three nonnaturally occurring CDR regions, and a light chain comprising three nonnaturally occurring CDR regions, wherein the six nonnaturally occurring CDRs are the CDRs of SEQ. ID. Nos. 28-33 respectively.

4. An antibody comprising a heavy chain comprising three nonnaturally occurring CDR regions, and a light chain comprising three nonnaturally occurring CDR regions, wherein the antibody specifically binds to human thrombomodulin, protein C, carbohydrate antigen, sialyl Lewis antigen, or electin.

5. An antibody comprising a heavy chain comprising three nonnaturally occurring CDR regions, and a light chain comprising three nonnaturally occurring CDR regions, wherein the antibody specifically binds to an antigen selected from the group consisting of bacterial LPS, virion capsid protein and envelope glycoprotein.

6. An antibody comprising a heavy chain comprising three nonnaturally occurring CDR regions, and a light chain comprising three nonnaturally occurring CDR regions, wherein the heavy and light chains are linked as a single chain via a linker peptide.

7. An antibody comprising a heavy chain comprising three nonnaturally occurring CDR regions, and a light chain comprising three nonnaturally occurring CDR regions, wherein the antibody specifically binds with an affinity of at least 10⁸ M⁻

1.