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