一种SUMO-Heparinase I融合酶的可溶性表达与纯化

Soluble Expression and Purification of the SUMO?鄄Heparanase I Fusion Enzyme

摘要:

克隆自肝素黄杆菌（Flavobacteriumheparinum）的肝素酶I（Heparinase I，EC4.2.2.7）广泛应用于低相对分子质量肝素（LMWH，low molecular weight heparin）制备研究。然而，该酶在大肠杆菌表达体系中易形成包涵体，限制了其大量应用。将可溶性配体SUMO（small ubiquitin?鄄like modifier，小泛素类似修饰蛋白质）与C端带有6×His标签的肝素酶基因片段N?鄄端融合表达，菌体总蛋白质和可溶性蛋白质SDS?鄄PAGE电泳表明，N端融合SUMO的肝素酶I可溶性表达比率显著提高；通过镍?鄄亲和柱层析得到均一纯化的融合肝素酶I，融合酶酶学性质表明该融合酶可直接应用于肝素降解而无需切除SUMO标签，为肝素酶I的广泛应用提供了良好的技术基础。

Abstract:

Heparinase I cloned from Flavobacterium heparinum was widely used in the preparation of LMWH（low molecular weight heparin）. However，the formation of inclusion bodies when expressed in Escherichia coli limits its application in a large scale. In this study，the soluble partner，small ubiquitin-like modifier（SUMO），was fused to the N-terminus of Hep I gene with a C-terminal 6×histidine tag. SDS-PAGE analysis of total protein in bacteria and soluble fractions indicated that the portion of the soluble Heparinase I fused with SUMO to the N-terminus was significantly enhanced. After purified by the nickel-chelate chromatography，the resulting fusion enzyme could be used directly without removing the fusion SUMO tag，which made the wide application of Heparinase I feasible.