重组GluV8的克隆表达、纯化及酶学性质研究  

Cloning,Expression,Purification,and Characterization of Recombinant GluV8

作　　者：段树燕 郭怀祖[2,3] 李晶 郑娟[1,2] 赵自叶 张大鹏[2,3] 王皓[3] 

机构地区：[1]聊城大学药学院,聊城252000 [2]抗体药物与靶向治疗国家重点实验室,上海201203 [3]第二军医大学肿瘤研究所,上海200433

出　　处：《中国生物工程杂志》2014年第4期36-40,共5页China Biotechnology

摘　　要：谷氨酰内切酶在生物制药及检测中应用较多,但来源受限,将全基因合成的金黄色葡萄球菌来源的谷氨酰内切酶功能区部分对应的基因进行改造后,克隆入表达载体pGEX-4T-2,导入E.coli BL21(DE3),重组蛋白以可溶性形式表达。采用亲和层析等纯化步骤对重组蛋白进行纯化,用底物Z-Phe-Leu-Glu-pNA(L-2135)对重组蛋白的酶学性质进行了研究,用HPLC、LC-MS/MS检测方法对酶切融合蛋白的位点特异性进行了鉴定。结果表明该酶的相对活性为1568U/mg,最适作用温度为42℃、最适作用pH为8.0,在pH 9.0,50℃时仍有较高的酶活,将该酶与胰酶酶切融合蛋白所得肽段结合能够提升质谱检测结果的精确度。以上结果表明该重组酶具有良好的应用前景。The glutamyl endopeptidase gene from Staphylococcus aureus was successfully expressed by cloning into the expression vector pGEX-4T-2 and then transformed into E. coil BI21 （DE3）. Then the recombinant GluV8 was purified by glutathione-Sepharose affinity column. For deamidating activity assay Z-Phe- Leu-Glu-pNA （L-2135） was used as substrate. The reaction result showed that recombinant GluV8 can effectively hydrolyze the alpha-carboxyl of glutamic acid residue, releasing p-nitroanilide with an activity of 1568U/rag. The optimal temperature and the pH is 42℃ and 8.0 respectively. And it remained most of activity at 50℃ and pH9.0. The specificity of recombinant GluV8 by HPLC and LC-MS/MS was identified. These results suggested that the recombinant protein is a relatively specific proteinase that could be effectively utilized for protein identification.

关 键 词：重组 GluV8 克隆 表达 酶学性质 

分 类 号：Q786[生物学—分子生物学]