重组hPRL-1的表达纯化及其理化和酶学性质研究  

Expression and Purification of Recombinant hPRL-1 and Its Physicochemical and Enzymatic Properties

作　　者：巴特[1] 

机构地区：[1]南开大学元素有机化学研究所元素有机化学国家重点实验室,天津300071

出　　处：《现代生物医学进展》2007年第12期1824-1828,共5页Progress in Modern Biomedicine

摘　　要：目的:表达纯化hPRL-1重组蛋白,分析其理化性质及酶学特性。方法:热激法将重组pET15b质粒转化入E.coli BL21中,IPTG诱导表达出His-tagged hPRL-1蛋白。使用Ni-NTA亲和层析法结合Mono Q离子交换层析法纯化。用SDS-PAGE法和Western Blot法进行表达情况的定性定量分析,并使用HPLC法鉴定蛋白纯度,计算出蛋白分子量,圆盘等电聚焦电泳分析重组蛋白等电点。比较分析以pNPP、4-MUP和DiFMUP为底物时的酶促反应动力学。同时以pNPP为底物测定酶的最适pH值;以4-MUP为底物测定酶的最适温度,分析探讨缓冲液离子强度与蛋白酪氨酸酶通用抑制剂钒酸钠对酶活力的影响。结果:以亲和层析和离子交换层析结合,可以纯化得到纯度约为95%的蛋白。测得蛋白分子量为24.54kD,等电点为9.11。以pNPP、4-MUP和DiFMUP为底物时Km分别为3720μmol/L,130μmol/L和50μmol/L。酶的最适pH值为7.6,最适温度为34℃。结论:纯化所得蛋白为目的蛋白hPRL-1;两步纯化相结合可以得到纯度较高的蛋白;三种底物特异性依次为DiFMUP>4-MUP>pNPP。Objective： To express, purify recombinant hPRL-1 in E, coli BL21, and analyze its chemical and enzymatic properties. Methods： Recombinant pET15b plasmid was transferred into E, coli BL21 to express His6-tagged hPRL-1, The Enzyme was purified by Ni-NTA IMAC strategy and Mono Q IEC strategy. SDS-PAGE and Western Blot were both used to evaluate the expression ofhPRL-1. The Molecular Weight was determined by HPLC, and the PI value was determined by IEF PAGE. Three kinds of substrates （pNPP, 4-MUP and DiFMUP） were used to determine the kinetics ofhPRL-1. Results： The purity ofhPRL-1 was about 95% aider the two-step purification. The Molecular Weight was 24.54kD and the PI value was 9.11. The Krn values for pNPP, 4-MUP and DiFMUP were respectively 3720μmol/L, 130μmol/L and 50μmol/L. The optimal pH value was 7.6, and the optimal temperature was about 34℃ Conclusions： Recombinant protein was obtained with high purity aider a two-step purification. The substrate specificity order is DiFMUP〉4-MUP〉DiFMUP.

关 键 词：hPLR-1 动力学 PNPP 4-MUP DiFMUP 

分 类 号：Q841[生物学]