Protein Design Automation for Protein Libraries

Abstract

The invention relates to the use of protein design automation (PDA) to generate computationally prescreened secondary libraries of proteins, and to methods and compositions utilizing the libraries.

1 . A method for generating a secondary library of scaffold protein variants comprising:
 providing a primary library comprising a filtered set of scaffold protein primary variant sequences;   generating a list of primary variant positions in said primary library;   combining a plurality of said primary variant positions to generate a secondary library of secondary sequences.   
   
   
       2 . A method for generating a secondary library of scaffold protein variants comprising:
 providing a primary library comprising a filtered set of scaffold protein primary variant sequences;   generating a probability distribution of amino acid residues in a plurality of variant positions;   combining a plurality of said amino acid residues to generate a secondary library of secondary sequences.   
   
   
       3 . A method according to  claim 1  further comprising synthesizing a plurality of said secondary sequences. 
   
   
       4 . A method according to  claim 2  wherein said synthesizing is done by multiple PCR with pooled oligonucleotides. 
   
   
       5 . A method according to  claim 4  wherein said pooled oligonucleotides are added in equimolar amounts. 
   
   
       6 . A method according to  claim 4  wherein said pooled oligonucleotides are added in amounts that correspond to the frequency of the mutation. 
   
   
       7 . A composition comprising a plurality of secondary variant proteins comprising a subset of said secondary library. 
   
   
       8 . A composition comprising a plurality of nucleic acids encoding a plurality of secondary variant proteins comprising a subset of said secondary library. 
   
   
       9 . A method for generating a secondary library of scaffold protein variants comprising:
 providing a first library filtered set of scaffold protein primary variants;   generating a probability distribution of amino acid residues in a plurality of variant positions;   synthesizing a plurality of scaffold protein secondary variants comprising a plurality of said amino acid residues to form a secondary library;   
     wherein at least one of said secondary variants is different from said primary variants.