| 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 |
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| 引用本文： | 傅俊华,王琪,尹杰超,刘铭瑶,李宁,姚文斌,任桂萍,李璐,李德山.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[J].生物工程学报,2010,26(6):837-842. |
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| 作者姓名： | 傅俊华 王琪 尹杰超 刘铭瑶 李宁 姚文斌 任桂萍 李璐 李德山 |
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| 作者单位： | 东北农业大学生命科学学院生物制药实验室,哈尔滨,150030 |
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| 基金项目： | 国家自然科学基金 (No. 30700591) 资助。 |
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| 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1.375×104U/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。
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| 关 键 词： | SUMO蛋白酶1(Ulp1)，谷胱甘肽S-转移酶 (GST)，高效表达，活性鉴定 |
| 收稿时间： | 1/8/2010 12:00:00 AM |
Expression and characterization of soluble recombinant Ulp1p with glutathione S-transferase tag in Escherichia coli |
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| Junhua Fu,Qi Wang,Jiechao Yin,Mingyao Liu,Ning Li,Wenbin Yao,Guiping Ren,Lu Li and Deshan Li.Expression and characterization of soluble recombinant Ulp1p with glutathione S-transferase tag in Escherichia coli[J].Chinese Journal of Biotechnology,2010,26(6):837-842. |
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| Authors: | Junhua Fu Qi Wang Jiechao Yin Mingyao Liu Ning Li Wenbin Yao Guiping Ren Lu Li and Deshan Li |
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| Institution: | Biopharmaceutical Laboratory, College of Life Science, Northeast Agricultural University, Harbin 150030, China;Biopharmaceutical Laboratory, College of Life Science, Northeast Agricultural University, Harbin 150030, China;Biopharmaceutical Laboratory, College of Life Science, Northeast Agricultural University, Harbin 150030, China;Biopharmaceutical Laboratory, College of Life Science, Northeast Agricultural University, Harbin 150030, China;Biopharmaceutical Laboratory, College of Life Science, Northeast Agricultural University, Harbin 150030, China;Biopharmaceutical Laboratory, College of Life Science, Northeast Agricultural University, Harbin 150030, China;Biopharmaceutical Laboratory, College of Life Science, Northeast Agricultural University, Harbin 150030, China;Biopharmaceutical Laboratory, College of Life Science, Northeast Agricultural University, Harbin 150030, China;Biopharmaceutical Laboratory, College of Life Science, Northeast Agricultural University, Harbin 150030, China |
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| 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. |
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| Keywords: | ubiquitin-like specific protease 1 (Ulp1) glutathione S-transferase (GST) high-level expression activity identification |
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