Methods and compositions for polypeptide engineering
View Patent ↗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.
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.