Expression, production, and characterization of full-length vitronectin in Escherichia coli
Expression, production, and characterization of full-length vitronectin in Escherichia coli
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DOI：
10.1016/j.pep.2004.04.004
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发表时间：
2004-07-01
影响因子：
1.6
通讯作者：
Hocking, DC
中科院分区：
文献类型：
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作者：
Wojciechowski, K;Chang, CH;Hocking, DC
Vitronectin(VN)是血清中的主要黏附蛋白之一，可促进多种细胞类型在组织培养塑料上的附着和扩散。本研究利用pGEX2t表达载体在大肠杆菌中以GST标记的融合蛋白的形式表达全长的人VN。用IPTG诱导GST/VN的产生，发现该蛋白定位于包涵体。从细胞裂解液中分离包涵体，用2M尿素和Triton X-100洗涤一次，然后在还原化合物存在的情况下用8M尿素溶解。用肝素亲和层析纯化溶解的GSTNN，用磷酸盐缓冲液透析复性。从1个L的细菌培养物中回收了约40 mg的GSTNN。纯化的GST/VN在SDS-PAGE上以预测的相对分子质量迁移，并可被抗GST和抗VN抗体识别。GST/VN与肝素结合，促进细胞黏附、铺展和生长，其程度与血浆来源的VN相似。因此，在细菌中生产重组VN是一种快速、方便地生产大量VN用于细胞研究的方法。(C)2004 Elsevier Inc.保留所有权利。
Vitronectin (VN) is one of the primary adhesive proteins in serum and serves to promote the attachment and spreading of a wide variety of cell types to tissue culture plastic. In this study, the pGEX2t expression vector was used to express full-length human VN as a GST-tagged fusion protein in Escherichia coli. GST/VN production was induced with IPTG and the protein was found to localize to inclusion bodies. The inclusion bodies were isolated from cell lysates, washed once with 2 M urea and Triton X-100, and then solubilized with 8 M urea in the presence of a reducing compound. Solubilized GSTNN was purified by heparin affinity chromatography and refolded by dialysis against phosphate buffered saline. Approximately 40 mg of GSTNN was recovered from 1 L of bacterial culture. Purified GST/VN migrated at the predicted molecular mass on SDS-PAGE and was recognized by both anti-GST and anti-VN antibodies. GST/VN bound to heparin and promoted cell adhesion, spreading, and growth to a similar extent as that observed with plasma-derived VN. As such, the production of recombinant VN in bacteria represents a rapid and convenient method to produce large quantities of VN for cellular studies. (C) 2004 Elsevier Inc. All rights reserved.