Analysis of the ligand binding properties of recombinant bovine liver-type fatty acid binding protein
Analysis of the ligand binding properties of recombinant bovine liver-type fatty acid binding protein
复制标题
DOI：
10.1016/0005-2760(95)00170-0
复制
发表时间：
1995-12-07
期刊：
影响因子：
--
通讯作者：
Spener, F
中科院分区：
文献类型：
--
作者：
Rolf, B;OudenampsenKruger, E;Spener, F
用RT-PCR方法扩增了牛肝型脂肪酸结合蛋白（L-FABP）的cDNA编码区，克隆了L-FABP基因，并将其用于大肠杆菌（E. coli）表达系统。重组蛋白的表达量占可溶性大肠杆菌的25%。大肠杆菌蛋白，并可以通过一个简单的两步协议结合离子交换层析和凝胶过滤分离。用滴定量热法测定了油酸、花生四烯酸、油酰辅酶A、溶血磷脂酸和过氧化物酶体增殖剂苯扎贝特与L-FABP结合的解离常数。所有配体以2：1的化学计量结合，第一配体结合的解离常数都在微摩尔范围内，油酸以最高的亲和力结合，Kd为0.26 μ M。此外，胆固醇与L-FABP的结合用Lipidex测定法、脂质体结合测定法和荧光置换测定法研究。在所有试验中均未观察到胆固醇与L-FABP的结合。
The coding part of the cDNA for bovine liver-type fatty acid binding protein (L-FABP) has been amplified by RT-PCR, cloned and used for the construction of an Escherichia coli (E. coli) expression system. The recombinant protein made up to 25% of the soluble E. coli proteins and could be isolated by a simple two step protocol combining ion exchange chromatography and gel filtration. Dissociation constants for binding of oleic acid, arachidonic acid, oleoyl-CoA, lysophosphatidic acid and the peroxisomal proliferator bezafibrate to L-FABP have been determined by titration calorimetry. All ligands were bound in a 2:1 stoichiometry, the dissociation constants for the first ligand bound were all in the micro molar range, Oleic acid was bound with the highest affinity and a K-d of 0.26 mu M. Furthermore, binding of cholesterol to L-FABP was investigated with the Lipidex assay, a liposome binding assay and a fluorescence displacement assay. In none of the assays binding of cholesterol to L-FABP was observed.