A highly effective and adjustable dual plasmid system for O-GlcNAcylated recombinant protein production in E-coli
A highly effective and adjustable dual plasmid system for O-GlcNAcylated recombinant protein production in E-coli
复制标题
一种高效且可调节的双质粒系统，用于在大肠杆菌中生产 O-GlcNAcylated 重组蛋白。
DOI：
10.1093/jb/mvv006
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发表时间：
2015-06-01
影响因子：
2.7
通讯作者：
Yu, Wengong
中科院分区：
文献类型：
--
作者：
Han, Cuifang;Shan, Hui;Yu, Wengong
O-GlcN酰化是后生动物中普遍存在的一种动态、可逆的蛋白质翻译后修饰，它分别由O-GlcNAc转移酶(OGT)和O-GlcNAcase催化和去除。原核生物缺乏内源性OGT活性。有报道称，哺乳动物OGT与其靶底物在大肠杆菌中共表达可产生O-GlcNacylated重组蛋白，但所用的载体不相容，OGT及其靶蛋白的表达都是由同一诱导剂诱导的。在这里，我们描述了一个兼容的双质粒系统，用于在大肠杆菌中共表达OGT及其目标底物，以生产O-GlcNacylated蛋白。以CKII和P53蛋白为对照，验证了该方法的有效性。这个相容的双质粒系统包含一个阿拉伯糖诱导的具有pUC起始点的OGT表达载体和一个具有P15A起始点的异丙基β-d-硫代半乳糖苷诱导型OGT靶底物表达载体。双质粒系统通过改变诱导剂浓度产生不同O-GlcN酰化水平的重组蛋白。更重要的是，O-GlcN酰化反应的效率比以前报道的体系要高得多。总之，我们建立了一个可调节的、相容的双质粒系统，该系统可以在大肠杆菌中有效地生产O-GlcNacylated蛋白。
O-GlcNAcylation is a ubiquitous, dynamic and reversible post-translational protein modification in metazoans, and it is catalysed and removed by O-GlcNAc transferase (OGT) and O-GlcNAcase, respectively. Prokaryotes lack endogenous OGT activity. It has been reported that coexpression of mammalian OGT with its target substrates in Escherichia coli produce O-GlcNAcylated recombinant proteins, but the plasmids used were not compatible, and the expression of both OGT and its target protein were induced by the same inducer. Here, we describe a compatible dual plasmid system for coexpression of OGT and its target substrate for O-GlcNAcylated protein production in E. coli. The approach was validated using the CKII and p53 protein as control. This compatible dual plasmid system contains an arabinose-inducible OGT expression vector with a pUC origin and an isopropyl β-d-thiogalactopyranoside-inducible OGT target substrate expression vector bearing a p15A origin. The dual plasmid system produces recombinant proteins with varying O-GlcNAcylation levels by altering the inducer concentration. More importantly, the O-GlcNAcylation efficiency was much higher than the previously reported system. Altogether, we established an adjustable compatible dual plasmid system that can effectively yield O-GlcNAcylated proteins in E. coli.