Sortase A酶突变体的原核表达、纯化及活性测定（英文）  

Prokaryotic Expression, Purification and Activity Determination of Sortase A Enzyme Mutant

作　　者：张庆彬 黄宗庆 路建光 赵文杰 冯军[1,2] ZHANG Qingbin;HUANG Zongqing;LU Jianguang;ZHAO Wenjie;FENG Jun(State Key Lab.of New Drug and Pharmaceutical Process,Shanghai Institute of Pharmaceutical Industry,China State Institute of Pharmaceutical Industry,Shanghai 201203;Shanghai Duomirui Bio-Technology Co.,Ltd.,Shanghai 201203)

机构地区：[1]中国医药工业研究总院上海医药工业研究院,创新药物与制药工艺国家重点实验室,上海201203 [2]上海多米瑞生物技术有限公司,上海201203

出　　处：《中国医药工业杂志》2020年第1期37-47,共11页Chinese Journal of Pharmaceuticals

基　　金：国家重点研发计划“新型动物药剂创制与产业化”项目(2017YFD0501404)

摘　　要：本研究采用pET28a载体对金黄色葡萄球菌(Staphylococcus aureus)中的转肽酶A(sortase A,SrtA)截短体SrtA?N25以及突变体m5SrtA?N59、m9SrtA?N59进行了原核发酵表达,并通过镍柱亲和色谱、阳离子交换色谱进行蛋白纯化。随后,通过荧光共振能量转移试验比较了3种酶的催化活性,测定了酶反应动力学常数、筛选了最适反应条件,并进一步探究了不同生化试剂对SrtA酶学活性的影响。结果显示,SrtA?N25、m5SrtA?N59、m9SrtA?N59在大肠埃希菌中均能以可溶形式高效表达,且纯化后得到的高纯度SrtA酶均具有生物学活性。相较于SrtA?N25,m5SrtA?N59、m9SrtA?N59活性分别提高了约68、5 544倍,尤其是m9SrtA?N59的催化活性大大提高。m9SrtA?N59的最适反应温度为30~37℃,最适反应pH值为6.0~7.0,反应体系中加入三(2-羧乙基)膦(TCEP)、二硫苏糖醇(DTT)等还原剂有助于增强SrtA酶催化活性。In this study, the SrtA?N25, a transpeptidase derived from Staphylococcus aureus, and its mutants(m5 SrtA?N59 and m9 SrtA?N59) were expressed in p ET28 a expression system, purified via Ni-Sepharose and cation-exchange chromatography subsequently. The enzyme activities, kinetic parameters and optimal reaction conditions of these enzymes were assessed on FRET-based platform, and effects of different biochemical reagents were evaluated on Srt A activity. As a result, SrtA?N25, m5 SrtA?N59 and m9 SrtA?N59 were expressed as a soluble form in Escherichia coli with a high yield and maintained enzyme activities. Finally, the Kcat/Km values of m5 SrtA?N59 and m9 SrtA?N59 respectively increased 68-fold and 5 544-fold compared to that of the SrtA?N25, suggesting the catalytic efficiency of m9 SrtA?N59 was improved significantly. The optimum reaction temperature of m9 SrtA?N59 was 30-37 ℃ and the optimum pH value was 6.0-7.0. Adding reducing agents such as tris-(2-carboxyethyl)phosphine(TCEP) and dithiothreitol(DTT) to the reaction system was helpful for enhancing the catalytic activity of Srt A enzyme.

关 键 词：金黄色葡萄球菌 转肽酶A 酶学活性 纯化 

分 类 号：Q814[生物学—生物工程] Q559