融合蛋白GST-Ulp1p在大肠杆菌中的高效可溶性表达及其活性鉴定

Expression and characterization of soluble recombinant Ulp1p with glutathione S-transferase tag in Escherichia coli

摘要:

利用基因工程技术，体外重组小分子类泛素修饰蛋白酶1 (Ulp1) 的活性片段，获得高表达、高特异性重组蛋白酶。从酿酒酵母Saccharomyces cerevisia中提取Ulp1编码第403到621个氨基酸残基之间的DNA片段 (Ulp1p)，在其C端加入6×His并连接到大肠杆菌表达载体pGEX中，构建重组表达质粒pGEX-Ulp1p-his6。将重组质粒转化至大肠杆菌Rosetta (DE3) 中，氨苄青霉素抗性筛选转化子。表达、纯化后，以SUMO融合蛋白检测其活性。经过优化，该蛋白可溶性表达，表达量占菌体总蛋白的40.12％。可通过谷胱甘肽琼脂糖凝胶柱或Ni-NTA凝胶亲和层析纯化得到纯度98％的蛋白。经酶切分析，比活力为1.375×104 U/mg。融合蛋白GST-Ulp1p-His6无需切除谷胱甘肽S-转移酶 (GST) 标签，具有很高的活性，制备简易；6×His标签，有利于底物蛋白切割后纯化，减少蛋白损失。本研究为制备高活力的SUMO蛋白酶提供了一个新方法。

Abstract:

The aim of the study is to obtain an efficient expression of recombinant ubiquitin-like specific protease 1 (Ulp1) by gene engineering. We cloned the Ulp1p, active fragment (403 aa?621 aa) of Ulp1, from Saccharomyces cerevisia, and subcloned into pGEX/Rosetta (DE3) to form an expression plasmid, pGEX-Ulp1p-His6. In order to enhance the solubility of GST-Ulp1p-His6, we purified the fusion protein GST-Ulp1p-His6 by either glutathione S-transferase agarose or Ni-NTA resin chromatography, the purity was up to 98%. We utilized the protein to cleave the SUMO fusions, and the specific activity of GST-Ulp1p-His6 was 1.375×104 U/mg. This study showed that the recombinant protein GST-Ulp1p-His6 displayed high specificity and activity.