Mutagenic Polymerase Chain Reaction of Protein-Coding Genes for In Vitro Evolution

In vitro protein evolution is an efficient approach to study the structure and function of a protein, or to enhance its industrial utility (1 ). One round of evolution consists of random mutation of a protein-coding gene, expression the resulting library in a population of micro-organisms, and high-throughput screening or selection of clones that most strongly exhibit a desired phenotype (“winners”). After many rounds, mutations that confer the phenotype accumulate on a single allele, e.g., the authors have isolated an octuple mutant of the Escherichia coli β-glucuronidase with catalytic activity resistant to roughly 80-fold higher concentrations of glutaraldehyde than that of the wild-type enzyme (2 ). Here we describe a variation of the mutagenic polymerase chain reaction (PCR) (3 ,4 ) is described. The advantages of this method over other random mutagenesis techniques are explained.