Velkov Tony, Chuang Sara, Prankerd Richard, Sakellaris Harry, Porter Christopher J H, Scanlon Martin J
Department of Medicinal Chemistry, Victorian College of Pharmacy, Monash University, 381 Royal Parade, Parkville, 3052 Victoria, Australia.
Protein Expr Purif. 2005 Nov;44(1):23-31. doi: 10.1016/j.pep.2005.04.006.
Rat liver fatty acid binding protein (L-FABP) was efficiently expressed in Escherichia coli and purified to homogeneity. The cDNA encoding L-FABP was ligated into the pTrc99A expression vector and expressed by induction with isopropyl-beta-d-thiogalactopyranoside under the control of the P(trc) promoter. Following an 18 h induction period, L-FABP constituted approximately 3% of the cytosolic protein. The protein could be purified to electrophoretic homogeneity (silver-stained polyacrylamide gel detection) by ammonium sulfate fractionation (65% saturation) of the soluble bacterial lysate followed by the chromatographic sequence of anion-exchange-->hydrophobic interaction-->anion-exchange chromatography. The recombinant protein displayed an isoelectric point of 7.0 and cross-reactivity with rabbit anti-(human L-FABP) polyclonal antibody. The ligand binding properties of the delipidated L-FABP were examined by titration with the fluorescent probe 1-anilino-8-naphthalene sulfonic acid and isothermal titration calorimetric analysis of oleic acid binding. The purified rat L-FABP displayed a binding stoichiometry of 2:1 (ANS:L-FABP) with dissociation constants (K(d)) of 1.7 and 15.5 microM for the high and low affinity binding sites, respectively. The K(d) values determined by ITC for oleic acid binding were 0.155 and 4.04 microM with a binding stoichiometry of approximately 2 mol of fatty acid/mol of protein. These physicochemical and binding properties are in agreement with those of L-FABP isolated from rat liver tissue.
大鼠肝脏脂肪酸结合蛋白（L-FABP）在大肠杆菌中高效表达并纯化至同质。将编码L-FABP的cDNA连接到pTrc99A表达载体中，并在P（trc）启动子的控制下用异丙基-β-D-硫代半乳糖苷诱导表达。经过18小时的诱导期，L-FABP约占胞质蛋白的3%。通过对可溶性细菌裂解物进行硫酸铵分级分离（65%饱和度），然后依次进行阴离子交换→疏水相互作用→阴离子交换色谱的层析序列，可将该蛋白纯化至电泳同质（银染聚丙烯酰胺凝胶检测）。重组蛋白的等电点为7.0，与兔抗（人L-FABP）多克隆抗体具有交叉反应性。通过用荧光探针1-苯胺基-8-萘磺酸滴定以及油酸结合的等温滴定量热分析，研究了脱脂L-FABP的配体结合特性。纯化的大鼠L-FABP与ANS的结合化学计量比为2:1（ANS:L-FABP），高亲和力和低亲和力结合位点的解离常数（K(d)）分别为1.7和15.5 microM。通过ITC测定的油酸结合的K(d)值为0.155和4.04 microM，脂肪酸与蛋白质的结合化学计量比约为2摩尔/摩尔。这些物理化学和结合特性与从大鼠肝脏组织中分离的L-FABP一致。
