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首页> 外文期刊>Protein Expression and Purification >Recombinant human SMOCs produced by in vitro refolding: Calcium-binding properties and interactions with serum proteins
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Recombinant human SMOCs produced by in vitro refolding: Calcium-binding properties and interactions with serum proteins

机译：体外重折叠产生的重组人SMOC：钙结合特性和与血清蛋白的相互作用

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Secreted modular calcium-binding (SMOC) proteins are little known members of the BM-40 family of matricellular proteins. SMOC-1 is localized in basement membranes, while SMOC-2 exhibits pro-angiogenic properties and stimulates cell cycle progression via integrin-linked kinase. in this work we have expressed recombinant human SMOCs in inclusion bodies in Escherichia coli. Soluble proteins were prepared by in vitro refolding with a final yield of approximately 3 mg of purified SMOCs per liter of bacterial culture. The folding state of the products and their ability to reversibly bind calcium ions were verified by CD spectroscopy. The EF hands of the refolded SMOCs were both functional, one had high affinity for calcium ions (K-d values in the 0.7-1 mu M range), while the other had lower affinity (K-d values 20-25 mu M). The proteins were also examined for their ability to bind blood serum proteins. Three of the bands specifically retained on SMOC-Sepharose were identified as C-reactive protein, an acute phase protein from the pentraxin family, the basement membrane and elastic fiber-associated fibulin-1, and vitronectin, which is involved in cell adhesion, migration and proliferation and binds numerous extracellular and membrane proteins, including integrins. The interactions were additionally confirmed in solution using purified individual proteins by the method of biotin label transfer from one interacting partner to the other. Their identification is among the first pieces of information about the action of the SMOCs on molecular level and opens new possibilities for future research aimed towards elucidating the physiological roles of these versatile proteins. (C) 2008 Elsevier Inc. All rights reserved.
机译：分泌的模块化钙结合（SMOC）蛋白是母细胞蛋白BM-40家族中鲜为人知的成员。 SMOC-1位于基底膜中，而SMOC-2具有促血管生成特性，并通过整合素连接的激酶刺激细胞周期进程。在这项工作中，我们已经在大肠杆菌的包涵体中表达了重组人SMOC。通过体外重折叠制备可溶性蛋白，最终产量约为每升细菌培养物3 mg纯化的SMOC。产品的折叠状态及其与钙离子可逆结合的能力已通过CD光谱法验证。重新折叠的SMOC的EF手均具有功能，一只对钙离子具有高亲和力（K-d值在0.7-1μM范围内），而另一只具有较低的亲和力（K-d值20-25μM）。还检查了蛋白质结合血清蛋白质的能力。特异性保留在SMOC-Sepharose上的三个带被鉴定为C反应蛋白，五色蛋白家族的急性期蛋白，基底膜和与弹性纤维相关的fibrin-1和玻连蛋白，它们参与细胞粘附，迁移。并与许多细胞外和膜蛋白结合，包括整联蛋白。还通过使用生物素标记从一种相互作用伴侣转移到另一种伴侣的方法，使用纯化的单个蛋白质在溶液中进一步确认了相互作用。它们的鉴定是关于SMOC在分子水平上作用的第一批信息，并且为旨在阐明这些多功能蛋白质的生理作用的未来研究提供了新的可能性。 （C）2008 Elsevier Inc.保留所有权利。

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