Rational engineering of a virulence gene from Mycobacterium tuberculosis facilitates proteomic analysis of a natural protein N-terminus.
Rational engineering of a virulence gene from Mycobacterium tuberculosis facilitates proteomic analysis of a natural protein N-terminus.
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结核分枝杆菌毒力基因的合理工程促进了天然蛋白质 N 末端的蛋白质组学分析。
DOI：
10.1038/srep33265
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发表时间：
2016
影响因子：
4.6
通讯作者：
Champion,PatriciaA
中科院分区：
文献类型：
--
作者：
Reyna,Cristal;MbaMedie,Felix;Champion,MatthewM;Champion,PatriciaA
用于检测蛋白质的质谱（MS）是用于评估蛋白质组的生物过程的不可或缺的工具。蛋白质组学经常需要将蛋白质水解成肽片段。蛋白质可能难以在序列水平上进行理想的蛋白质水解，使得它们难以通过常规蛋白质组学方法进行分析。EsxA（ESAT-6，Early Secreted Antigen，6 kDa）是结核分枝杆菌（Mycobacterium tuberculosis）的主要毒力决定因子。EsxA通常用于在实验室中评估分枝杆菌毒力，并作为人类结核病的生物标志物。EsxA的序列阻碍了常规检测之外的更深入的MS分析。在这里，我们工程EsxA的序列，以增加理想的胰蛋白酶的性质，旨在改善复杂的MS分析。我们证明EsxA变体适合MS分析，并且在已建立的Esx-1功能体外和体内测定中仍然具有功能。我们提供了分子工程的第一个示范，以专门提高个别微生物蛋白质的MS分析。
Mass spectrometry (MS) for the detection of proteins is an indispensable tool for evaluating the biological processes of the proteome. Proteomics frequently requires proteolysis of proteins into peptide fragments. Proteins can be refractory to ideal proteolysis at the sequence level rendering them difficult to analyze by routine proteomics methods. EsxA (ESAT-6, Early Secreted Antigen, 6kDa) is a major virulence determinant ofMycobacterium tuberculosis,the cause of human tuberculosis. EsxA is routinely used to evaluate mycobacterial virulence in the laboratory and as a biomarker for tuberculosis in humans. The sequence of EsxA hinders deeper MS analysis beyond routine detection. Here we engineer the sequence of EsxA to add desirable tryptic properties aimed at improving complex MS analysis. We demonstrate that EsxA variants are amenable to MS analysis and remain functional in establishedin vitroandex vivoassays of Esx-1-function. We provide the first demonstration of molecular engineering to specifically improve MS analysis of individual microbial proteins.