An Enhanced System for Unnatural Amino Acid Mutagenesis in E. coli
An Enhanced System for Unnatural Amino Acid Mutagenesis in E. coli
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DOI：
10.1016/j.jmb.2009.10.030
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发表时间：
2010-01-15
影响因子：
5.6
通讯作者：
Schultz, Peter G.
中科院分区：
文献类型：
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作者：
Young, Travis S.;Ahmad, Insha;Schultz, Peter G.
我们报告了一种新的载体，pEVOL，用于在大肠杆菌中使用进化的詹氏甲烷球菌氨酰-tRNA合成酶（s）（阿尔斯）/抑制tRNA对将非天然氨基酸掺入蛋白质中。这种新的系统通过使用组成型和诱导型启动子来驱动两个拷贝的M.詹氏阿尔斯基因通过在单载体构建体中将双aaRS启动子系统与新优化的抑制tRNA（CUA）（opt）偶联，产量进一步增加。与先前报道的用于非天然氨基酸诱变的载体相比，优化的抑制tRNA（CUA）（opt）提供了增加的质粒稳定性。为了证明这个新系统的实用性，我们引入了14个突变阿尔斯pEVOL和比较他们的能力，插入非天然氨基酸在抹香鲸肌红蛋白或绿色荧光蛋白中的三个独立的琥珀无义密码子。当在摇瓶中在丰富培养基中培养时，pEVOL能够产生超过100 mg/L的突变GroEL蛋白。pEVOL载体的多功能性、增加的产率和增加的稳定性将进一步促进具有非天然氨基酸的蛋白质的表达。(C)2009爱思唯尔有限公司保留所有权利。
We report a new vector, pEVOL, for the incorporation of unnatural amino acids into proteins in Escherichia coli using evolved Methanocaldococcus jannaschii aminoacyl-tRNA synthetase(s) (aaRS) /suppressor tRNA pairs. This new system affords higher yields of mutant proteins through the use of both constitutive and inducible promoters to drive the transcription of two copies of the M. jannaschii aaRS gene. Yields were further increased by coupling the dual-aaRS promoter system with a newly optimized suppressor tRNA(CUA)(opt) in a single-vector construct. The optimized suppressor tRNA(CUA)(opt) afforded increased plasmid stability compared with previously reported vectors for unnatural amino acid mutagenesis. To demonstrate the utility of this new system, we introduced 14 mutant aaRS into pEVOL and compared their ability to insert unnatural amino acids in response to three independent amber nonsense codons in sperm whale myoglobin or green fluorescent protein. When cultured in rich media in shake flasks, pEVOL was capable of producing more than 100 mg/L mutant GroEL protein. The versatility, increased yields, and increased stability of the pEVOL vector will further facilitate the expression of proteins with unnatural amino acids. (C) 2009 Elsevier Ltd. All rights reserved.