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

被引：42

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

来源：

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS | 2012年 / 418卷 / 04期

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

页码：652 / 656

页数：5

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