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

被引:41
作者
Ozawa, Kiyoshi [1 ,2 ]
Loscha, Karin V. [1 ]
Kuppan, Kekini V. [1 ]
Loh, Choy Theng [1 ]
Dixon, Nicholas E. [2 ]
Otting, Gottfried [1 ]
机构
[1] Australian Natl Univ, Res Sch Chem, Canberra, ACT 0200, Australia
[2] Univ Wollongong, Sch Chem, Wollongong, NSW 2522, Australia
基金
澳大利亚研究理事会;
关键词
Aminoacyl-tRNA-synthetase; Cell-free protein synthesis; Escherichia coli; Suppressor tRNA; Unnatural amino acids; GENERAL-METHOD; DNA; POLYMERASE; EXPRESSION; BINDING;
D O I
10.1016/j.bbrc.2012.01.069
中图分类号
Q5 [生物化学]; Q7 [分子生物学];
学科分类号
071010 ; 081704 ;
摘要
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. (C) 2012 Elsevier Inc. All rights reserved.
引用
收藏
页码:652 / 656
页数:5
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