Efficient Sampling of Protein Sequence Space for Multiple Mutants

Abstract

We describe here a method capable of generating a very large population of multiple mutants, the size of which is primarily limited by volume constraints. This method, referred to as recombination-enhanced mutagenesis, combines the power of in vitro mutagenesis with the high frequencies of in vivo recombination that can be achieved using single-stranded transduction systems. The recombination frequency between two mutations separated by as little as 19 amino acids is 0.02; this frequency approaches a value of 0.1 for mutations separated by more than 38 amino acids. Up to 10(8) independent recombinants were generated in 1 ml of an E. coli culture, and this number scales linearly (or better) with increasing volume. To prove the method's effectiveness, we applied it to the problem of reverting multiple mutants of mouse dihydrofolate reductase, which could not be reverted using mutagenesis alone. Thus, given an appropriate screen or selection scheme, recombination-enhanced mutagenesis is well-suited for addressing a range of combinatorially complex problems, such as antigen recognition, enzyme catalysis, protein folding, and transport/transduction across biomembranes.

Evolutionally guided enzyme design

“…The method involves the use of transducing phage to mobilize a pool of plasmid-borne mutations into cells containing a comparably diverse mutant pool (Caren et al, 1994). Our results demonstrate that the recombination frequency between any two mutations separated by as little as 100 bp (i.e., approximately 35 amino acids) approaches 10%.…”