Efficient synthesis of nonnatural mutants in Escherichia coli S30 in vitro protein synthesizing system

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.