Peeters R A, Ena J M, Veerkamp J H
Department of Biochemistry, University of Nijmegen, The Netherlands.
Biochem J. 1991 Sep 1;278 ( Pt 2)(Pt 2):361-4. doi: 10.1042/bj2780361.
The coding part of the cDNA encoding human muscle fatty-acid-binding protein (FABP) was ligated in the pET8c vector and expressed under the control of the lacUV5 promoter. After induction with isopropyl beta-D-thiogalactopyranoside, almost 12% of the cytoplasmic proteins consisted of FABP. The protein could be isolated after sonication of the bacterial pellet followed by (NH4)2SO4 precipitations, anion-exchange chromatography and gel filtration. The muscle FABP produced in Escherichia coli has an isoelectric point of 5.3 and is recognized by anti-(human muscle FABP) antiserum after Western blotting. The purified FABP has a preference for binding to palmitic acid and C18-C22 (poly)unsaturated fatty acids, and no affinity to palmitoyl-CoA or other hydrophobic ligands tested. The dissociation constant for oleic acid is 0.58 microM, with a binding stoichiometry of 0.72 mol of fatty acid/mol of protein. The physicochemical and binding characteristics of the protein were in complete agreement with those of FABP isolated from human skeletal muscle.
编码人肌肉脂肪酸结合蛋白（FABP）的cDNA编码部分被连接到pET8c载体中，并在lacUV5启动子的控制下表达。用异丙基β-D-硫代半乳糖苷诱导后，近12%的细胞质蛋白由FABP组成。细菌沉淀经超声处理，然后进行硫酸铵沉淀、阴离子交换色谱和凝胶过滤后，可分离出该蛋白。在大肠杆菌中产生的肌肉FABP的等电点为5.3，经蛋白质印迹法后可被抗（人肌肉FABP）抗血清识别。纯化的FABP优先结合棕榈酸和C18 - C22（多）不饱和脂肪酸，对棕榈酰辅酶A或其他测试的疏水配体无亲和力。油酸的解离常数为0.58微摩尔，脂肪酸与蛋白质的结合化学计量比为0.72摩尔脂肪酸/摩尔蛋白质。该蛋白的物理化学和结合特性与从人骨骼肌中分离的FABP完全一致。
