Methods and compositions for polypeptide engineering
AssigneeMaxygen, Inc.
InventorsPhillip Patten, Willem Stemmer
Life of this patent
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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.
Claims14
A method for evolving a protein encoded by a DNA substrate molecule comprising:
- (a) digesting at least a first and second DNA substrate molecule, wherein the at least a first and second substrate molecules differ from each other in at least one nucleotide, with a restriction endonuclease;
- (b) ligating the mixture to generate a library of recombinant DNA molecules;
- (c) screening or selecting the products of (b) for a desired property;
- and
- (d) recovering a recombinant DNA substrate molecule encoding an evolved protein.
-15. (canceled)
A method for evolving a protein encoded by a DNA substrate molecule by recombining at least a first and second DNA substrate molecule, wherein the at least a first and second substrate molecules differ from each other in at least one nucleotide and comprise defined segments, the method comprising:
- (a) providing a set of oligonucleotide PCR primers, comprising at least one primer for each strand of each segment, wherein the primer sequence is complementary to at least one junction with another segment;
- (b) amplifying the segments of the at least a first and second DNA substrate molecules with the primers of step (a) in a polymerase chain reaction;
- (c) assembling the products of step (b) to generate a library of recombinant DNA substrate molecules;
- (d) screening or selecting the products of (c) for a desired property;
- and
- (e) recovering a recombinant DNA substrate molecule from (d) encoding an evolved protein.
-194. (canceled)
A method for evolving an alpha interferon gene comprising:
- (a) providing a library of mutant alpha interferon genes, the library comprising a filamentous phage vector;
- (b) stimulating cells comprising a reporter construct, the reporter construct comprising a reporter gene under control of an interferon responsive promoter, and wherein the reporter gene is GFP;
- (c) separating the cells expressing GFP by FACS;
- (d) recovering phage from the product of (c);
- and
- (e) recovering an evolved interferon gene from the product of (d).
(canceled)
-201. (canceled)
-216. (canceled)
A method for evolving a DNA substrate molecule encoding an interferon alpha, comprising:
- (a) providing a library of mutant alpha interferon genes, the library comprising an expression vector wherein the alpha interferon genes are expressed under the control of an inducible promoter;
- (b) transfecting host cells with the library of (a);
- (c) contacting the product of (b) with a virus;
- (d) recovering DNA encoding a mutant alpha interferon from host cells surviving step (c);
- and
- (e) recovering an evolved interferon gene from the product of (d).
-273. (canceled)
Dependencies are inferred from the claim text.