High yield, purity and activity of soluble recombinant Bacteroides thetaiotaomicron GST-heparinase I from Escherichia coli
High yield, purity and activity of soluble recombinant Bacteroides thetaiotaomicron GST-heparinase I from Escherichia coli
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DOI：
10.1016/j.abb.2007.01.029
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发表时间：
2007-04-01
影响因子：
3.9
通讯作者：
McKeehan, Wallace L.
中科院分区：
文献类型：
--
作者：
Luo, Yongde;Huang, Xinqiang;McKeehan, Wallace L.
肝黄杆菌中的肝素酶I是多种多糖酶的来源，但在缺乏糖胺聚糖多糖酶的大肠杆菌中以重组产物表达时，存在产率低、纯度不足、不溶性等问题。本研究利用哺乳动物肠道共生体拟杆菌（Bacteroides thelaiotaomicron）的基因组信息构建了F. heparinum肝素酶I同源基因的cDNA，并在大肠杆菌中以n端GST融合蛋白的形式表达。这导致了高产量（30 mg/g干菌）的可溶性产物，并促进了一步亲和纯化到均匀性。以肝素（平均12,000 Da）为底物，经GST融合纯化的肝素酶I的K-m为2.3 μ M， V-max为42.7 μ Mol/min，比活性为164 U/mg。结果表明，在产量，比活性和亲和力的肝素作为底物比以往的报道2倍的提高。这些数据表明，来自肠道共生体的肝素酶I对肝素的内在亲和力高于来自肝素F的肝素酶I。纯化后的GST融合酶需要Ca2+， pH值在6.7 ~ 7.3之间，与去除n端GST部分的酶相似。我们的研究表明，肝素酶I的催化活性需要还原环境。GST促进了肝素酶I在固相中的固定化，无论是用于临床目的还是用于结构研究，都不会受到污染多糖酶的干扰。(c) 2007爱思唯尔公司版权所有。
Heparinase I from Flavobacterium heparinum, a source of diverse polysaccharidases, suffers from low yields, insufficient purity for structural studies and insolubility when expressed as a recombinant product in Escherichia coli that is devoid of glycosaminoglycan polysaccharidases. In this study, cDNA coding for the orthologue of F. heparinum heparinase I was constructed from genomic information from the mammalian gut symbiont Bacteroides thelaiotaomicron and expressed in E. coli as a fusion protein with GST at the N-terminus. This resulted in high yield (30 mg/g dry bacteria) of soluble product and facilitated one-step affinity purification to homogeneity. Purified heparinase I bearing the GST fusion exhibited a K-m of 2.3 mu M and V-max of 42.7 mu Mol/min with a specific activity of 164 U/mg with heparin (average 12,000 Da) as substrate. The results indicate a 2-fold improvement in yield, specific activity and affinity for heparin as substrate over previous reports. The data suggest that the heparinase I from the gut symbiont exhibits a higher intrinsic affinity for heparin than that from F heparinum. The purified GST fusion enzyme exhibited a requirement for Ca2+ and a pH optimum between 6.7 and 7.3 that was similar to the enzyme freed of the N-terminal GST portion. Our study revealed that catalytic activity of heparinase I requires a reducing environment. The GST facilitated immobilization of heparinase I in solid phase either for clinical purposes or for structural studies in absence of interference by contaminating polysaccharidases. (c) 2007 Elsevier Inc. All rights reserved.