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.

1 . A method for protein engineering comprising the steps of: 
 a) providing a library of protein variants;    b) testing said library of protein variants for at least one property of interest in a test of interest;    c) identifying a range of values for said at least one property of interest;    d) identifying a minimum within said range of values that is associated with a favorable outcome in said test of interest; and    e) providing a plurality of protein variants having at least one mutation above said minimum in said range of said at least one property of interest, thereby providing a library of protein variants comprising at least one mutation, and wherein said library is enriched in members having said favorable outcome in said test of interest.    
     
     
         2 . The method of  claim 1 , wherein said favorable outcome corresponds to a value of greater than 50%, 60%, 70%, 80%, 90%, or 95% of a maximal value observed in said test of step (a).  
     
     
         3 . The method of  claim 1 , wherein said protein is an enzyme.  
     
     
         4 . The method of  claim 2 , wherein said enzyme is selected from proteases, transferases, metalloproteases, esterases, amylases, cellulases, oxidases, cutinases, and lipases.  
     
     
         5 . A method for protein engineering comprising the steps of: 
 a) providing a library of protein variants;    b) testing said library of protein variants for at least two properties of interest in a test of interest;    c) identifying a range of values for said at least two properties of interest;    d) identifying a minimum within said range of values that is associated with a favorable outcome in said test of interest; and    e) providing a plurality of protein variants above said minimum of said range of said at least two properties of interest, thereby providing a library of protein variants enriched in members having said favorable outcome in said test of interest.    
     
     
         6 . The method of  claim 5 , wherein said favorable outcome corresponds to a value of greater than 50%, 60%, 70%, 80%, 90%, or 95% of a maximal value observed in said test of step (a).  
     
     
         7 . The method of  claim 5 , wherein said protein is an enzyme.  
     
     
         8 . The method of  claim 7 , wherein said enzyme is selected from proteases, transferases, metalloproteases, esterases, amylases, cellulases, oxidases, cutinases, and lipases.  
     
     
         9 . A method for protein engineering comprising the steps of: 
 a) providing a wild-type protein and a library of protein variants of said wild-type protein;    b) testing said library of protein variants and said wild-type protein for at least one property of interest in a test of interest;    c) identifying a range of values for said at least one property of interest;    d) identifying a minimum within said range of values that is associated with a favorable outcome in said test of interest;    e) identifying said protein variants having a favorable outcome as compared to the results obtained for said wild-type, wherein said favorable outcome is an improved property of interest; and    f) providing a plurality of protein variants above said minimum of said range of said at least one property of interest, thereby providing a library of improved protein variants enriched in members having said favorable outcome in said test of interest.    
     
     
         10 . The method of  claim 9 , further comprising the step of determining the performance index, wherein said performance index is determined by dividing the value obtained for each of said improved protein variants and the value obtained for said wild-type protein.  
     
     
         11 . The method of  claim 9 , further comprising the step of identifying said improved protein variants, wherein said improved protein variants achieve performance index values greater than 1.1 in said test of interest.  
     
     
         12 . The method of  claim 9 , wherein said protein is an enzyme.  
     
     
         13 . The method of  claim 12 , wherein said enzyme is selected from proteases, transferases, metalloproteases, esterases, amylases, cellulases, oxidases, cutinases, and lipases.  
     
     
         14 . The method of  claim 9 , wherein said protein is selected from antibodies and growth factors.  
     
     
         15 . The method of  claim 9 , wherein said wild-type protein is a mature form an enzyme selected from proteases, transferases, metalloproteases, esterases, amylases, cellulases, oxidases, cutinases, and lipases.  
     
     
         16 . The method of  claim 9 , wherein said property of interest is 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.  
     
     
         17 . The method of  claim 9 , wherein said wild-type and said protein variant are components of at least one detergent composition.  
     
     
         18 . The method of  claim 16 , 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.  
     
     
         19 . A method for producing an improved variant of a parent protein within a protein fold, comprising: 
 a) assaying multiple variants of a test protein within said protein fold spanning a range of a property of interest in an assay of interest;    b) identifying a minimum within said range of said property of interest that is associated with a favorable outcome in said assay of interest;    c) assaying a parent protein of said protein fold in said assay of interest; and    d) producing an improved variant of said parent protein by introducing an amino acid substitution is said parent protein such that said improved variant is above said minimum of said range of said property of interest.    
     
     
         20 . The method of  claim 19 , wherein said parent protein and said test protein are different.  
     
     
         21 . The method of  claim 19 , further comprising the step of determining the performance index, wherein said performance index is determined by dividing the value obtained for said improved protein variant and the value obtained for said parent protein.  
     
     
         22 . The method of  claim 19 , wherein said test proteins and said parent proteins are enzymes.  
     
     
         23 . The method of  claim 22 , wherein said enzymes are selected from proteases, transferases, metalloproteases, esterases, amylases, cellulases, oxidases, cutinases, and lipases.  
     
     
         24 . The method of  claim 23 , wherein said parent protein is a mature form an enzyme selected from proteases, transferases, metalloproteases, esterases, amylases, cellulases, oxidases, cutinases, and lipases.  
     
     
         25 . The method of  claim 19 , wherein said property of interest is 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.  
     
     
         26 . The method of  claim 19 , wherein said test protein and said parent protein are components of at least one detergent composition.