title: Efficient synthesis of nonnatural mutants in Escherichia coli S30 in vitro protein synthesizing system.
abstract: Factors that affect the efficiency of in vitro synthesis of mutant proteins that contain nonnatural amino acids were investigated. The process of the nonnatural mutagenesis consists of chemical aminoacylation of a tRNA that contains a 4-base anticodon, followed by in vitro synthesis in the presence of an mRNA that contains the corresponding 4-base codon. Detailed studies on the time courses of the synthesis revealed two major factors that suppress the yield of nonnatural mutants compared with the wild-type protein. First, a cyclic tRNA that exists as a by-product of the chemical aminoacylation inhibits the protein synthesis. Second, the very short lifetime of a tRNA aminoacylated with a nonnatural amino acid limits the protein yield. As a simple and practical way of surmounting these factors, aminoacyl tRNA was added into the in vitro system at 5 min after the start of the synthesis. The addition increased the protein yield up to the level of conventional proteins in the in vitro system.
authors.affiliation: Department of Bioscience and Biotechnology, Faculty of Engineering, Okayama University, 3-1-1 Tsushimanaka, Okayama 700-8530, Japan.
authors.forename: Katsuhiro
authors.initials: K
authors.lastname: Yamanaka
authors.forename: Hidetaka
authors.initials: H
authors.lastname: Nakata
authors.forename: Takahiro
authors.initials: T
authors.lastname: Hohsaka
authors.forename: Masahiko
authors.initials: M
authors.lastname: Sisido