Method for In Vitro Molecular Evolution of Protein Function

Abstract

The present invention relates to a method for in vitro creation of molecular libraries evolution of protein function. Particularly, it relates to variability and modification of protein function by shuffling polynucleotide sequence segments. A protein of desired characteristics can be obtained by incorporating variant peptide regions (variant motifs) into defined peptide regions (scaffold sequence). The variant motifs can be obtained from parent DNA which has been subjected to mutagenesis to create a plurality of differently mutated derivatives thereof or they can be obtained from in vivo sequences. These variant motifs can then be incorporated into a scaffold sequence and the resulting coded protein screened for desired characteristics. This method is ideally used for obtaining antibodies with desired characteristics by isolating individual CDR DNA sequences and incorporating them into a scaffold which may, for example, be from a totally different antibody.

1 - 19 . (canceled) 
     
     
         20 . A method of creating a polynucleotide library, each polynucleotide encoding an antibody or a part thereof, comprising the steps of:
 (a) obtaining a parent polynucleotide encoding an antibody or part thereof having one or more variant protein motifs;   (b) providing a plurality of pairs of oligonucleotides, each pair representing spaced apart locations on the parent polynucleotide sequence bounding an intervening variant polynucleotide sequence encoding a variant protein motif, and using each said pair of oligonucleotides as amplification primers for PCR to amplify the intervening variant polynucleotide sequence encoding a variant protein motif;   (c) isolating the nucleotide sequences amplified in step (b);   (d) obtaining single-stranded nucleotide sequences from the isolated amplified nucleotide sequences from step (c);   (e) assembling polynucleotide sequences by incorporating the single-stranded nucleotide sequences derived from step (d) with nucleotide sequences encoding scaffold sequence; and   (f) inserting the assembled polynucleotide sequences of step (e) into suitable vectors thereby forming said polynucleotide library, wherein the assembled polynucleotide sequence encodes an antibody or part thereof.   
     
     
         21 . The method according to  claim 20  further comprising the step of expressing said polynucleotide sequences to obtain corresponding library of antibodies or parts thereof. 
     
     
         22 . The method according to  claim 21  further comprising screening the library for an antibody or part thereof of desired characteristics. 
     
     
         23 . A method of creating an antibody or part thereof having desired characteristics comprising,
 (a) creating a polynucleotide library according to  claim 20 , and   (b) selecting and expressing said polynucleotide sequences to obtain an antibody or part thereof of desired characteristics.   
     
     
         24 . The method according to  claim 20  wherein the assembled polynucleotide sequence encodes an antibody part selected from the group consisting of an antigen binding fragment (Fab), a fragment consisting of a variable domain of heavy chain and heavy chain constant region 1 (Fd), a variable fragment (Fv), a variable domain of heavy chain (dAb), a pepsin cleavage fragment comprising two Fab fragments and a hinge region (F(ab′) 2 ), a single-chain variable fragment (scFv), and an isolated complementarity determining region (CDR). 
     
     
         25 . The method according to  claim 22  wherein said antibody or part thereof of desired characteristics is an antibody part selected from the group consisting of an antigen binding fragment (Fab), a fragment consisting of a variable domain of heavy chain and heavy chain constant region 1 (Fd), a variable fragment (Fv), a variable domain of heavy chain (dAb), a pepsin cleavage fragment comprising two Fab fragments and a hinge region (F(ab′) 2 ), a single-chain variable fragment (scFv), and an isolated complementarity determining region (CDR). 
     
     
         26 . The method according to  claim 23  wherein said antibody or part thereof of desired characteristics is an antibody part selected from the group consisting of an antigen binding fragment (Fab), a fragment consisting of a variable domain of heavy chain and heavy chain constant region 1 (Fd), a variable fragment (Fv), a variable domain of heavy chain (dAb), a pepsin cleavage fragment comprising two Fab fragments and a hinge region (F(ab′) 2 ), a single-chain variable fragment (scFv), and an isolated complementarity determining region (CDR). 
     
     
         27 . The method according to  claim 20 , wherein one of said pair of oligonucleotides is linked to a member of a specific binding pair (MSBP). 
     
     
         28 . The method according to  claim 27 , wherein step (c) comprises binding the MSBP of the amplified polynucleotide sequence to its specific binding partner. 
     
     
         29 . The method according to  claim 27 , wherein the MSBP is biotin. 
     
     
         30 . The method according to  claim 28 , wherein the specific binding partner is streptavidin. 
     
     
         31 . The method according to  claim 20 , wherein the parent polynucleotide sequence is subjected to error prone PCR prior to step (b).