Streptavidin binding and endothelial cell adhesion to biotinylated fibronectin
Streptavidin binding and endothelial cell adhesion to biotinylated fibronectin
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DOI：
10.1021/la702322n
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发表时间：
2007-12-04
期刊：
影响因子：
3.9
通讯作者：
Reichert, William M.
中科院分区：
文献类型：
--
作者：
Anamelechi, Charles C.;Clermont, Edward E.;Reichert, William M.
将高亲和力的链霉亲和素-生物素结合和低亲和力的纤维连接蛋白-整合素配体结合在一起的双配体系统被开发出来，以增强内皮细胞与聚合物的黏附。在这项研究中，我们研究了生物素化纤维连接蛋白(BfN)作为对先前开发的DL方法的增强的实用性。其目标是通过消除异种牛血清白蛋白(BBSA)使该系统更适合临床研究。用Sulfo-NHS-LC-Biotin实现纤维连接蛋白(FN)的生物素化。利用表面等离子体共振(SPR)光谱技术，研究了结合生物素与野生型链霉亲和素(WT-SA)和突变株链霉亲和素(RGD-SA)的亲和力。酶联免疫吸附分析(EL ISA)吸光度值证实，与未修饰的天然蛋白相比，细胞结合结构域在轻度生物素化的BfN上是可访问的。SPR结合分析证实了WT-SA和RGD-SA与BfN的结合行为相似。然而，动力学分析表明，由于每个FN的生物素增加，亲和力没有增加，这表明系统中缺乏正的协作性。我们通过SPR验证了BfN在亲和结合中的重要作用，并通过ELISA法证实了整合素-Fn连接的可能性。最后，使用纽蛋白免疫染色来确定在DL系统中使用BfN形成局灶性粘连。在DL系统中，使用BFN的病灶粘连密度明显高于单独使用FN。
A dual ligand (DL) system that combines high affinity streptavidin-biotin binding with lower affinity fibronectin-integrin ligand binding was developed to augment endothelial cell adhesion to polymers. In this study, we examined the utility of biotinylated fibronectin (bFN) as an enhancement to the previously developed DL approach. The goal was to make the system more amenable to clinical studies by eliminating xenogenic bovine serum albumin (bBSA). Fibronectin (FN) biotinylation was achieved with Sulfo-NHS-LC-Biotin. The affinity of conjugated biotin for wildtype streptavidin (WT-SA) and a mutant strain streptavidin (RGD-SA) was measured using surface plasmon resonance (SPR) spectroscopy. Enzyme-Linked ImmunoSorbent Assay (ELISA) absorbance values confirmed the accessibility of the cell binding domain on mildly biotinylated bFN when compared to unmodified native protein. SPR binding analysis confirmed similar binding behavior to bFN with WT-SA and RGD-SA. Kinetic analysis, however, showed no increase in affinity due to increased biotins per FN, an indication of the absence of positive cooperativity in the system. We verified the essential utility of bFN in affinity binding by SPR and confirmed the potential for integrin-FN linkages by ELISA. Finally, Vinculin immunostaining was used to determine focal adhesion formation using bFN in the DL system. Significantly greater focal adhesion density was achieved with the bFN in the DL system than with FN alone.