Screening of amber suppressor tRNAs suitable to introduce nonnatural amino acids into proteins by real-time monitoring of cell-free translation.

Abstract

Incorporation of nonnatural amino acids into proteins is a useful technique to analyze protein structure and function. We have reported that amber suppressor tRNAs suitable for efficient and specific incorporation of nonnatural amino acids into proteins can be obtained by screening a wide variety of naturally occurring tRNAs in an E. coli. cell-free translation system. The amber suppressor activity of the tRNAs was evaluated by incorporation of a fluorescent nonnatural amino acid and fluorescent SDS-PAGE analysis of cell-free translation products, though the SDS-PAGE was troublesome and time-consuming. In this research, we developed an alternative method for the screening of amber suppressor tRNAs by real-time measurement of fluorescence resonance energy transfer (FRET) from GFP to BODIPY558-linked nonnatural amino acid, which was incorporated into the N-terminus of GFP by amber suppressor tRNAs. Using this method, we demonstrated that the screening of the amber suppressor activity of various prokaryotic Trp tRNAs was performed in a high-throughput manner.