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 - 55 . (canceled) 
     
     
         56 . A method of generating an assembled polynucleotide sequence encoding a protein of desired characteristics comprising the steps of:
 a) providing at least one polynucleotide sequence comprising one or more variant polynucleotide sequences encoding one or more in vivo formed variant protein motifs;   b) providing one or more pairs of defined oligonucleotides, each pair representing spaced apart locations on the at least one polynucleotide sequence of step a), each pair binding adjacent to a variant polynucleotide sequence encoding an in vivo formed variant protein motif;   c) using the pairs of defined oligonucleotides as amplification primers for PCR to amplify the variant polynucleotide sequences encoding the in vivo formed variant protein motifs of the at least one polynucleotide sequence of step a) and performing PCR amplification on the at least one polynucleotide sequences of step a);   d) obtaining one or more single-stranded polynucleotide sequences from the amplified polynucleotide sequences in step c);   e) providing one or more unmutated, specifically selected, scaffold polynucleotide sequences encoding one or more mutated peptide regions; and   f) annealing said one or more single-stranded polynucleotide sequences from step d) with the unmutated, specifically selected, scaffold polynucleotide sequences from step e) such that annealed polynucleotides with one or more gaps is formed, and filling the one or more gaps present in the annealed polynucleotides, thereby generating one or more assembled polynucleotide sequences.