PDF(Pubmed)
Abstract：
Complications related to atherosclerosis account for approximately 1 in 4 deaths in the United States and treatment has focused on lowering serum LDL-cholesterol levels with statins. However, approximately 50% of those diagnosed with atherosclerosis have blood cholesterol levels within normal parameters. Human fortilin is an anti-apoptotic protein and a factor in macrophage-mediated atherosclerosis and is hypothesized to protect inflammatory macrophages from apoptosis, leading to subsequent cardiac pathogenesis. Fortilin is unique because it provides a novel drug target for atherosclerosis that goes beyond lowering cholesterol and utilization of a solution nuclear magnetic resonance (NMR) spectroscopy, structure-based drug discovery approach requires milligram quantities of pure, bioactive, recombinant fortilin. Here, we designed expression constructs with different affinity tags and protease cleavage sites to find optimal conditions to obtain the quantity and purity of protein necessary for structure activity relationship studies. Plasmids encoding fortilin with maltose binding protein (MBP), 6-histidine (6His) and glutathione-S-transferase (GST), N- terminal affinity tags were expressed and purified from Escherichia coli (E. coli). Cleavage sites with tobacco etch virus (TEV) protease and human rhinovirus (HRV) 3C protease were assessed. Despite high levels of expression of soluble protein, the fusion constructs were resistant to proteinases without the inclusion of amino acids between the cleavage site and N-terminus. We surveyed constructs with increasing lengths of glycine/serine (GGS) linkers between the cleavage site and fortilin and found that inclusion of at least one GGS insert led to successful protease cleavage and pure fortilin with conserved binding to calcium as measured by NMR.
摘要：
在美国，与动脉粥样硬化有关的并发症约占四分之一的死亡，并且治疗集中于使用他汀类药物降低血清LDL-胆固醇水平。然而,诊断为动脉粥样硬化的患者中约有50％的血液胆固醇水平在正常参数范围内。人fortilin是一种抗凋亡蛋白和巨噬细胞介导的动脉粥样硬化的因子，被认为可以保护炎性巨噬细胞免受凋亡。导致随后的心脏发病机制。Fortilin是独一无二的，因为它为动脉粥样硬化提供了一种新颖的药物靶标，超越了降低胆固醇和利用溶液核磁共振（NMR）光谱，基于结构的药物发现方法需要毫克量的纯，生物活性，重组fortilin。这里,我们设计了具有不同亲和标签和蛋白酶切割位点的表达构建体，以找到最佳条件，以获得结构活性关系研究所需的蛋白质数量和纯度。编码具有麦芽糖结合蛋白（MBP）的长子蛋白的质粒，6-组氨酸（6His）和谷胱甘肽-S-转移酶（GST），从大肠杆菌(E.大肠杆菌）。评估了烟草蚀刻病毒（TEV）蛋白酶和人鼻病毒（HRV）3C蛋白酶的切割位点。尽管高水平的可溶性蛋白表达，融合构建体对蛋白酶具有抗性,而在切割位点和N末端之间不包含氨基酸。我们调查了在切割位点和fetilin之间具有增加长度的甘氨酸/丝氨酸（GGS）接头的构建体，发现包含至少一个GGS插入物导致成功的蛋白酶切割和纯fetilin，通过NMR测量与钙保守结合。
