Improved Incorporation of Noncanonical Amino Acids by an Engineered tRNATyr Suppressor

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• 作者：Benjamin J. Rauch ; Joseph J. Porter ; Ryan A. Mehl ; John J. Perona
• 刊名：Biochemistry
• 出版年：2016
• 出版时间：January 26, 2016
• 年：2016
• 卷：55
• 期：3
• 页码：618-628
• 全文大小：502K
• ISSN：1520-4995

文摘

The Methanocaldcoccus jannaschii tyrosyl-tRNA synthetase (TyrRS):tRNA^Tyr cognate pair has been used to incorporate a large number of noncanonical amino acids (ncAAs) into recombinant proteins in Escherichia coli. However, the structural elements of the suppressor tRNA^Tyr used in these experiments have not been examined for optimal performance. Here, we evaluate the steady-state kinetic parameters of wild-type M. jannaschii TyrRS and an evolved 3-nitrotyrosyl-tRNA synthetase (nitroTyrRS) toward several engineered tRNA^Tyr suppressors, and we correlate aminoacylation properties with the efficiency and fidelity of superfolder green fluorescent protein (sfGFP) synthesis in vivo. Optimal ncAA-sfGFP synthesis correlates with improved aminoacylation kinetics for a tRNA^Tyr amber suppressor with two substitutions in the anticodon loop (G34C/G37A), while four additional mutations in the D and variable loops, present in the tRNA^Tyr used in all directed evolution experiments to date, are deleterious to function both in vivo and in vitro. These findings extend to three of four other evolved TyrRS enzymes that incorporate distinct ncAAs. Suppressor tRNAs elicit decreases in amino acid K_m values for both TyrRS and nitroTyrRS, suggesting that direct anticodon recognition by TyrRS need not be an impediment to superior performance of this orthogonal system and offering insight into novel approaches for directed evolution. The G34C/G37A tRNA^Tyr may enhance future incorporation of many ncAAs by engineered TyrRS enzymes.