High-yield cell-free protein synthesis for site-specific incorporation of unnatural amino acids at two sites

Authors

Kiyoshi Ozawa, University of WollongongFollow
Karin V. Loscha, Australian National University
Kekini V. Kuppan, Australian National University
Choy Theng Loh, Australian National University
Nicholas E. Dixon, University of WollongongFollow
Gottfried Otting, Australian National University

RIS ID

53052

Publication Details

Ozawa, K., Loscha, K. V., Kuppan, K. V., Loh, C. Theng., Dixon, N. E., Otting, G. (2012). High-yield cell-free protein synthesis for site-specific incorporation of unnatural amino acids at two sites. Biochemical and Biophysical Research Communications, 418 (4), 652-656.

Abstract

Using aminoacyl-tRNA synthetase/suppressor tRNA pairs derived from Methanocaldococcus jannaschii, an Escherichia coli cell-free protein production system affords proteins with site-specifically incorporated unnatural amino acids (UAAs) in high yields through the use of optimized amber suppressor tRNA_CUA opt and optimization of reagent concentrations. The efficiency of the cell-free system allows the incorporation of trifluoromethyl-phenylalanine using a polyspecific synthetase evolved previously for p-cyanophenylalanine, and the incorporation of UAAs at two different sites of the same protein without any re-engineering of the E. coli cells used to make the cell-free extract.