A new feasible method for fibrinogen purification based on the affinity of Staphylococcus aureus clumping factor A to fibrinogen
A new feasible method for fibrinogen purification based on the affinity of Staphylococcus aureus clumping factor A to fibrinogen
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DOI：
10.1016/j.pep.2008.05.007
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发表时间：
2008-09-01
影响因子：
1.6
通讯作者：
Chang, Ling-Ya
中科院分区：
文献类型：
--
作者：
Liu, Chao-Zong;Cheng, Hui-Ju;Chang, Ling-Ya
血浆纤维蛋白原参与多种生理和病理过程，是生物医学研究的重要材料。在这里，我们报告了一种新的方便的方法，纤维蛋白原纯化的基础上的亲和性金黄色葡萄球菌凝集因子A的纤维蛋白原。成簇因子A（Clumping factor A，ClfA）是一种锚定在S.通过包含残基221-550的片段以高亲和力与纤维蛋白原γ链C末端结合的金黄色细菌。ClfA（ClfA（221-550））的这种活性通过重组技术与谷胱甘肽-S-转移酶（GST）的C-末端融合产生，并用作捕获血浆纤维蛋白原的亲和配体。将GST-ClfA（221-550）融合蛋白通过其GST部分固定在填充在塑料柱中的谷胱甘肽缀合的珠上。然后，用柠檬酸化和肝素化人血浆加载该亲和柱。在洗去未结合的蛋白质后，用柠檬酸盐缓冲溶液（50 mM，pH 5.6）特异性洗脱柱捕获的纤维蛋白原。纯化的人纤维蛋白原具有支持血小板粘附和聚集的能力，并通过凝血酶形成纤维蛋白凝块，表明ClfA（221-550）纯化的人纤维蛋白原是一种功能活性产物。我们还发现，无论是大鼠和小鼠纤维蛋白原可以纯化，以及人纤维蛋白原与此方法。ClfA（221-550）法简便易行，对需要纤维蛋白原的研究者有很大的帮助。(C)2008年爱思唯尔公司All rights reserved.
Plasma fibrinogen participates in several physiological and pathological events thus becoming a useful studying material in biomedical research. Here we report a new convenient method for fibrinogen purification based on the affinity of Staphylococcus aureus clumping factor A to fibrinogen. Clumping factor A (ClfA) is a cell wall-anchored surface protein of S. aureus bacteria that binds with a high affinity to the fibrinogen gamma chain C-terminus via a segment encompassing the residues 221-550. This activity of ClfA (ClfA(221-550)) was produced in fusion to the C-terminus of glutathione-S-transferase (GST) with recombinant technology and used as an affinity ligand to capture plasma fibrinogen. GST-ClfA(221-550) fusion protein was immobilized onto the glutathione-conjugated beads packed in a plastic column by its GST part. Then, this affinity column was loaded with citrated and heparinized human plasma. After washing out unbound proteins, column-captured fibrinogen was specifically eluted down with a citrate buffer solution (50 mM, pH 5.6). Purified human fibrinogen exhibited the ability to support platelet adhesion and aggregation and formed fibrin clot by thrombin, indicating that ClfA(221-550)-purified human fibrinogen is a functionally active product. We also found that both the rat and mouse fibrinogens could be purified as well as human fibrinogen with this method. By virtue of its simplicity and feasibility, ClfA(221-550)-based method would be very useful to the investigators who need fibrinogen to perform their studies. (C) 2008 Elsevier Inc. All rights reserved.