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.
Kanno
Takuya
T
Cell-Free Science and Technology Research Center and Venture Business Laboratory, Ehime University, Ehime, Japan.
Tozawa
Yuzuru
Y
D000818:Animals; D002474:Cell-Free System; D004798:Enzymes / Q000096:biosynthesis* / Q000235:genetics; D057166:High-Throughput Screening Assays; D006801:Humans; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D016133:Polymerase Chain Reaction; D014176:Protein Biosynthesis; D015202:Protein Engineering / Q000379:methods*; D012333:RNA, Messenger / Q000096:biosynthesis; D012639:Seeds / Q000201:enzymology; D013379:Substrate Specificity; D013997:Time Factors; D014158:Transcription, Genetic; D014908:Triticum / Q000196:embryology / Q000201:enzymology* / Q000235:genetics
D004798:Enzymes; D012333:RNA, Messenger