Methods and compositions for polypeptide engineering

• US 7,776,598 B2
• Filed: 02/05/2008
• Issued: 08/17/2010
• Est. Priority Date: 12/18/1996
• Status: Expired due to Fees

• Current Assignee: Codexis, Inc.
• Original Assignee: Maxygen Inc.
• Inventors: Patten, Phillip A., Stemmer, Willem P. C.
• Primary Examiner(s): WHISENANT, ETHAN C
• Application Number:
  US12/069,011
• Publication Number:
  US 20090149331A1
• Time in Patent Office: 924 Days
• Field of Search: 435/440, 435/6, 536/23.1, 530/350
• US Class Current: 435/440
• CPC Class Codes: A61K 38/45 Transferases (2)
• CPC Class Codes: A61K 48/00 Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
• CPC Class Codes: C07K 14/43595 from coelenteratae, e.g. medusae
• CPC Class Codes: C07K 14/545 IL-1
• CPC Class Codes: C07K 14/56 IFN-alpha
• 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 15/10 Processes for the isolation, preparation or purification of DNA or RNA chemical preparation of DNA or RNA C07H21/00; preparation of non-structural polynucleotides from microorganisms or with enzymes C12P19/34
• 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 15/1034 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 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 15/67 General methods for enhancing the expression
• CPC Class Codes: C12N 2795/14043 viral genome or elements thereof as genetic vector
• CPC Class Codes: C12N 2795/14143 viral genome or elements thereof as genetic vector
• CPC Class Codes: C12N 7/00 Viruses; Bacteriophages; Compositions thereof; Preparation or purification thereof preparing medicinal viral antigen or antibody compositions, e.g. virus vaccines, A61K39/00
• CPC Class Codes: C12N 9/0069 : acting on single donors with incorporation of molecular oxygen, i.e. oxygenases (1.13)
• CPC Class Codes: C12N 9/1007 : Methyltransferases (general) (2.1.1.)
• CPC Class Codes: C12N 9/16 : acting on ester bonds (3.1)
• CPC Class Codes: C12N 9/86 : acting on amide bonds in cyclic amides, e.g. penicillinase (3.5.2)
• CPC Class Codes: C12Q 1/6811 : Selection methods for production or design of target specific oligonucleotides or binding molecules
• CPC Class Codes: C12Q 1/6853 : using modified primers or templates
• CPC Class Codes: C12Q 1/686 : Polymerase chain reaction [PCR]
• 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
• CPC Class Codes: G01N 33/68 : involving proteins, peptides or amino acids involving lipoproteins G01N33/92
• CPC Class Codes: G01N 33/6845 : Methods of identifying protein-protein interactions in protein mixtures

Abstract

Methods are provided for the evolution of proteins of industrial and pharmaceutical interest, including methods for effecting recombination and selection. Compositions produced by these methods are also disclosed.

Claims

1. A method for evolving a protein having at least two subunits, comprising:

a) providing a library of mutant DNA substrate molecules for each subunit;

b) recombining the libraries into a library of single-chain constructs of the protein, the single chain construct comprising a DNA substrate molecule encoding each subunit sequence, the subunit sequence being linked by a linker at a nucleic acid sequence encoding the amino terminus of one subunit to a nucleic acid sequence encoding the carboxy terminus of a second subunit;

c) screening or selecting the products of (b);

d) recovering recombinant single chain construct DNA substrate molecules from the products of (c);

e) subjecting the products of (d) to mutagenesis; and

f) recovering an evolved single chain construct DNA substrate from (e).

2. The method of claim 1, wherein the products of (b) are displayed on a phage.

3. The method of claim 1, wherein the protein is selected from Table I.

4. The method of claim 1, wherein (a)-(f) are repeated.

5. The method of claim 1, wherein the products of (f) are subjected to mutagenesis.

6. The method of claim 1, wherein mutagenesis comprises recursive sequence recombination.

7. The method of claim 1, wherein the products of claim 1, are used in (a).

8. The method of claim 1, wherein the evolved DNA substrate molecule of (f) comprises a library of DNA substrate molecules.