Systematic evaluation of sequence and activity relationships using site evaluation libraries for engineering multiple properties

Abstract

The present invention provides methods for protein engineering. Specifically, the invention provides methods utilizing site evaluation libraries to design libraries that optimize two or more properties of a protein.

What is claimed is: 
     
         1 - 26 . (canceled) 
     
     
         27 . A method for protein engineering variants of a parent protein comprising the steps of:
 a) providing a parent protein and a site evaluation library of protein variants of said parent protein, wherein the site evaluation library comprises variants of the parent protein modified at one site of interest;   b) testing said library of protein variants and said parent protein for at least two properties of interest in respective tests of interest;   c) determining a performance index value for said at least two properties of interest by dividing the value obtained for the protein variants and the value obtained for said parent protein in the test of interest to provide an apparent ΔΔG app  for the protein variants compared to said parent protein in the test of interest;   d) determining a predicted performance index value for protein variants that combine mutations at two or more sites of interest, wherein said two or more sites of interest include productive sites that have at least one substitution that is better than the parent protein and does not include unproductive sites that have no substitutions better than the parent protein, wherein the predicted performance index value for the variants that combine two or more mutations are obtained by adding the ΔΔG app  values for those mutations; and   e) identifying from the predicted performance index value obtained in step (d), the protein variants that combine two or more mutations and which possess a first property that is improved relative to the parent protein and a second property that is at least 90% of that of the parent protein, thereby providing a library of protein variants enriched in members that have at least two properties of interest.   
     
     
         28 . The method of  claim 27 , wherein the properties of interest are selected from charge, wash performance, hard surface cleaning performance, thermal stability, storage stability, detergent stability, substrate binding, enzyme inhibition, expression level, reaction rate, and substrate degradation. 
     
     
         29 . The method of  claim 27 , wherein said protein is an enzyme. 
     
     
         30 . The method of  claim 29 , wherein said enzyme is selected from proteases, transferases, metalloproteases, esterases, amylases, cellulases, oxidases, cutinases, and lipases. 
     
     
         31 . The method of  claim 27 , wherein said protein is selected from antibodies and growth factors. 
     
     
         32 . The method of  claim 27 , wherein said parent protein and said protein variant are components of at least one detergent composition. 
     
     
         33 . The method of  claim 28 , wherein said wash performance is tested in a detergent composition formulated into a powdered or liquid detergent having a pH of between 5 and 12.0. 
     
     
         34 . The method of  claim 27 , wherein said parent protein and said protein variant are components of at least one detergent composition. 
     
     
         35 . The method of  claim 27 , further comprising selecting protein variants having a performance index greater than 1.1 for at least one property of said at least two properties of interest. 
     
     
         36 . The method of  claim 27 , further comprising selecting protein variants having a value greater than 50% of a maximal value set in said test of interest for at least one property of said at least two properties of interest.