Using E. coli-based cell-free protein synthesis to evaluate the kinetic performance of an orthogonal tRNA and aminoacyl-tRNA synthetase pair

► The orthogonal synthetase derived from M. jannaschii is an inefficient catalyst. ► The estimated turnover number for the orthogonal synthetase is 5.4 × 10−5 s−1. ► The o-tRNA concentration in the standard cell-free platform is 0.6 ± 0.1 μM. ► In vivo- and in vitro-produced orthogonal tRNA seem equally competent.