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

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Abstract

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-β-d-thiogalactopyranoside under the control of the Ptrc 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 (Kd) of 1.7 and 15.5 μM 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 μM 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.

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Materials

DNA polymerase, 1-anilino-8-naphthalene sulfonic acid (ANS) and oleic acid were obtained from Sigma (Sydney, NSW, Australia). E. coli strain BL21 Codon Plus (DE3)-RIL was purchased from Stratagene (La Jolla, CA). All other reagents were of the highest purity commercially available.

Cloning of rat FABP

L-FABP cDNA was generated by reverse-transcription of rat intestinal mRNA with the L-FABP-specific primer HS4 (5′-AAAAGGATCCTGTAAAAGAATATGAAATACAGCC-3′). The L-FABP gene fragment was subsequently amplified from the

Expression of the rat L-FABP in E. coli

The cDNA fragment encoding the complete rat L-FABP sequence was cloned into the pTrc99A plasmid downstream of the hybrid trp/lac promoter, Ptrc, to allow for inducible and efficient intracellular expression of rat L-FABP in E. coli (Fig. 1). Induction of expression was verified by Western blotting and was not detectable in uninduced cells (Fig. 2). L-FABP was detected in the soluble fraction of the E. coli extract and was not detectable in the cell wall fraction or media, indicating the protein

Discussion

L-FABP has previously been expressed in E. coli and isolated [9], [18], [19], [20], [21]. However, the published purification protocol yielded a heterogeneous protein mixture containing contamination with an endogenous E. coli protein. Although this protein does not appear to significantly affect the measured affinities of high affinity ligands such as oleic acid and ANS, our interests are in characterizing affinities of much lower affinity ligands. Therefore, it is necessary to have a highly

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