Creation of diversity in polypeptides

Abstract

The inventors realized that the diversity generated by conventional methods may be limited by steric hindrance between amino acid residues in the three-dimensional structures of the resulting polypeptides. The steric hindrance may occur between amino acid residues at widely different positions in the amino acid sequences, e.g. between residues in two different domains of the 3D structure, and resulting polypeptides which include such steric hindrance may never be observed in the conventional recombination methods because they may be ex-pressed in poor yields or may have poor activity or stability. The inventors developed a method to identify and alleviate such steric hindrance in the resulting polypeptides. In an alignment of the three-dimensional structures, steric hindrance is indicated when residues from two different structures are located within a certain distance. Pairs of residues at corresponding positions in the amino acid sequences are not considered, and residues close to the surface (high solvent accessibility) are considered to be less prone to steric hindrance.

1 - 13 . (canceled)  
     
     
         14 . A method of constructing a polypeptide, comprising: 
 a) structurally aligning a three-dimensional structure of a first parent polypeptide and a three-dimensional structure of a second parent polypeptide,    b) selecting a first amino acid residue from the structure of the first polypeptide and a second amino acid residue from the structure of the second polypeptide, such that: 
 i) the two selected residues are not aligned with each other in the structural alignment,  
 ii) a non-hydrogen atom of the first amino acid residue and a non-hydrogen atom of the second amino acid residue are located less than 2.7 Å apart, and  
 iii) each of the two residues is not Glycine and has a side chain having less than 30% solvent accessibility, and  
   c) substituting or deleting the first and/or the second residue with a smaller residue, and    d) recombining the amino acid sequences after the substitution, and    e) preparing a DNA-sequence encoding the polypeptide of step e) and expressing the polypeptide in a transformed host organism.    
     
     
         15 . The method of  claim 14 , wherein the parent polypeptides have an amino acids sequence identity of at least 50% to each other.  
     
     
         16 . The method of  claim 14 , wherein the parent polypeptides have an amino acids sequence identity of at least 80% to each other.  
     
     
         17 . The method of  claim 14 , wherein the parent polypeptides have an amino acids sequence identity of at least 90% to each other.  
     
     
         18 . The method of  claim 14 , wherein the parent polypeptides have an amino acids sequence homology of at least 50% to each other.  
     
     
         19 . The method of  claim 14 , wherein the parent polypeptides have an amino acids sequence homology of at least 80% to each other.  
     
     
         20 . The method of  claim 14 , wherein the parent polypeptides have an amino acids sequence homology of at least 90% to each other.  
     
     
         21 . The method of  claim 14 , further comprising 
 f) superimposing the structures so as to align each non-hydrogen atom located <10 Å of an atom in the first or the second residue, and    g) selecting two residues that are less than 1.5 Å apart in the new superimposition.    
     
     
         22 . The method of  claim 14  wherein the two selected residues after the substitution can form a hydrogen bond, a salt bridge or a cysteine bridge.  
     
     
         23 . The method of  claim 14  wherein a non-hydrogen atom of the first residue and a non-hydrogen atom of the second residue are located less than 1.5 Å apart.  
     
     
         24 . The method of  claim 14  wherein a non-hydrogen atom of the first residue and a non-hydrogen atom of the second residue are located less than 1.2 Å apart.  
     
     
         25 . The method of  claim 14  wherein a non-hydrogen atom of the first residue and a non-hydrogen atom of the second residue are located less than 1.1 Å apart.  
     
     
         26 . The method of  claim 14  wherein a non-hydrogen atom of the first residue and a non-hydrogen atom of the second residue are located less than 1.0 Å apart.  
     
     
         27 . The method of  claim 14  wherein each parent polypeptide is an enzyme having an active site, and the structural alignment is done so as to align each non-hydrogen atom of the amino acid residues of the active sites  
     
     
         28 . The method of  claim 14  wherein the enzymes belong to glycosyl hydrolase family  13 .  
     
     
         29 . The method of  claim 14  wherein the enzymes are a cyclodextrin glucanotransferase and a maltogenic alpha-amylase.  
     
     
         30 . The method of  claim 21  which further comprises producing a polypeptide having the recombined amino acid sequence, testing the polypeptide for an enzymatic activity and selecting an enzymatically active polypeptide.  
     
     
         31 . A polypeptide which has an amino acid identity of at least 80% to SEQ ID NO: 18 or 19 or 20 or 21 or 22 or 23 or 24 or 25.  
     
     
         32 . A polypeptide which: 
 a) has an amino acid sequence which is a hybrid of a maltogenic alpha-amylase and a cyclodextrin glucanotransferase,    b) has a smaller residue at a position corresponding to: 
 i) D209, L261, D267, M307, H503, T509, V626, K651 of SEQ ID NO: 6 or  
 ii) K7, Y181, N266, K270, L286, Y574, P592, S676 of SEQ ID NO: 17; and  
   c) has hydrolytic activity on starch.    
     
     
         33 . A dough comprising the polypeptide of  claim 32.