An Improved method for the purification of rat liver-type fatty acid binding protein from Escherichia coli

T. Velkov, S. Chuang, R. Prankerd, Harry Sakellaris, C.J.H. Porter, M.J. Scanlon

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    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-p-D-thiogalactopyranoside under the control of the P, 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 anionexchange -> 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 sulfuric 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 mu M for the high and low affinity binding sites, respectively. The Kd values determined by ITC for oleic acid binding were 0.155 and 4.04 mu M 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. (c) 2005 Elsevier Inc. All rights reserved.
    Original languageEnglish
    Pages (from-to)23-31
    JournalProtein Expression and Purification
    Issue number1
    Publication statusPublished - 2005

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