鸡氨肽酶H在大肠杆菌中的表达、纯化与部分酶学性质分析  

Expression in Escherichia coli,Purification and Enzymatic Properties of Chicken Aminopeptidase H

作　　者：赖庆安[1] 刘树滔[1] 卢菀华[1] 陈莉[1] Toshihide NISHIMURA 饶平凡[1] 

机构地区：[1]福州大学生物科学与工程学院,生物工程研究所,福州350001 [2]Graduate School of Biosphere Science, Hiroshima University, Japan

出　　处：《生物工程学报》2008年第3期381-386,共6页Chinese Journal of Biotechnology

基　　金：南昌大学食品科学教育部重点实验室开放基金(No.NCU200403)~~

摘　　要：氨肽酶H(Aminopeptidase H,APH))是生物组织内一种常见的氨基内肽酶,但因为天然材料中含量很低,基本无法深入研究其催化机理、功能与结构的关系及其在生物体内的确切功能。从鸡肝组织克隆了APH的全基因序列,并把该序列亚克隆到载体pET22b(+)上,然后转化大肠杆菌Rosetta(DE3),构建了APH的表达菌株。该菌株经IPTG诱导,在SDS-PAGE上明显出现一条与天然APH理论分子量一致的新增蛋白带,该条带的浓度随着表达时间的延长逐渐加深;6h基本达到平衡,此时重组蛋白占总蛋白的16.7%,表达水平高达94.7mg/L。对表达产物进行了活性检测、纯化和酶学性质分析,发现重组蛋白在亚基构成,热稳定性,最适pH等方面与天然APH基本相同,据此可以确认表达产物确实是APH,发酵液总活力达到1636u/L。这些结果为APH的催化机理及其在生物体内的功能的阐明奠定了重要的物质基础。Aminopeptidase H （APH） is an universally distributed aminoendopeptidase in the tissue of many organism. However, it is hard to investigate its mechanism underlying the catalysis and the function in cell. In this paper, the full DNA sequence of this enzyme was cloned from chicken liver, then subcloned to the vector pET22 b（＋）. The recombined vector was transformed into E. coli Rosetta（DE3）, and the APH gene was expressed by the induction of IPTG. It was found the recombinant protein exhibited same molecular weight as authentic APH on SDS-PAGE analysis; the expression level increased with induction time and approached maximum of 94,7mg/L till 6 hours, which contained 16.7% of the total protein, Moreover, this recombinant protein showed similar properties of subunit composition, thermal stability and optimum pH with native APH, based on the enzymatic assay, purification and analysis of enzymological properties, Therefore, it is confirmed that APH was expressed in this prokaryote system with a high-level of 1636 u/L aminopeptidase activity, These results would help to elucidate the catalysis mechanism and biological function of APH by providing enough material.

关 键 词：氨肽酶H(APH) 表达 纯化 酶活 

分 类 号：Q78[生物学—分子生物学] TE626.86[石油与天然气工程—油气加工工程]