Sortase A酶突变体的原核表达、纯化及活性测定（英文）

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。

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

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.