An improved system for the generation and analysis of mutant proteins containing unnatural amino acids in Saccharomyces cerevisiae
An improved system for the generation and analysis of mutant proteins containing unnatural amino acids in Saccharomyces cerevisiae
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DOI：
10.1016/j.jmb.2007.05.017
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发表时间：
2007-08-03
影响因子：
5.6
通讯作者：
Brock, Ansgar
中科院分区：
文献类型：
--
作者：
Chen, Shawn;Schultz, Peter G.;Brock, Ansgar
我们之前已经描述了一种方法，可以在原核生物和真核生物中对多种非天然氨基酸进行遗传编码。在这里，我们报道了一个系统优化的酿酒酵母表达系统，用于生产含有非天然氨基酸的突变蛋白。的表达水平显著增加。大肠杆菌琥珀色抑制因子tRNA（CUA）和同源的氨基酰基tRNA合成酶（aaRS）对，并通过强转录启动子、优化密码子和提高质粒拷贝数来表达靶蛋白基因。在这个新体系中，许多非天然氨基酸，包括光交联剂对苯甲酰苯丙氨酸和对氮杂苯丙氨酸，以及化学反应性氨基酸，对乙酰苯丙氨酸和对丙氧基苯丙氨酸，以良好的产量（在大多数情况下，最高可达6-8 mg/l）加入酵母的人类超氧化物歧化酶（hSOD）中。hSOD突变体的质谱分析在高动态范围内进行，使用多种反应监测，为控制非天然氨基酸掺入保真度的因素提供了新的见解。(c) 2007 Elsevier Ltd.版权所有。
We have previously described methodology that makes it possible to genetically encode a wide array of unnatural amino acids in both prokaryotic and eukaryotic organisms. Here, we report the systematic optimization of a Saccharomyces cerevisiae expression system for the production of mutant proteins containing unnatural amino acids. Modifications include significant increases in both the expression levels of. the orthogonal Escherichia coli amber suppressor tRNA(CUA) and cognate aminoacyl-tRNA synthetase (aaRS) pair, and expression of the target protein gene using a strong transcriptional promoter, optimized codons and elevated plasmid copy numbers. With this new system, a number of unnatural amino acids, including the photocrosslinkers p-benzoylphenylalanine and p-azidophenylalanine, and the chemically reactive amino acids, p-acetylphenylalanine and p-propargyloxyphenylalanine, were incorporated into human superoxide dismutase (hSOD) in yeast in good yields (maximally similar to 6-8 mg/l of culture in most cases). Mass spectrometric analysis of the hSOD mutants was performed with high dynamic range using multiple reaction monitoring that provided new insights into the factors that control the fidelity of unnatural amino acid incorporation. (c) 2007 Elsevier Ltd. All rights reserved.