Protein engineering accelerated by cell-free technology.

Utilization of structural information from homologous proteins to design novel enzymes is one of the practical applications of structural biology. Structure-based protein engineering is a more reasonable strategy compared with general random mutagenesis. Here, we describe a useful method for production of a series of mutant enzymes based on a cell-free translation system. We employed PCR-mediated in vitro site-directed mutagenesis in combination with wheat-embryo cell-free protein synthesis to establish a high-throughput system. The efficient generation of a series of mutant enzymes facilitates high-throughput screening of functionally improved enzymes.