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Expression of Human Liver Fatty Acid-Binding Protein in Escherichia Coli and Comparative Analysis of Its Binding Characteristics With Muscle Fatty Acid-Binding Protein

Expression of Human Liver Fatty Acid-Binding Protein in Escherichia Coli and Comparative Analysis of Its Binding Characteristics With Muscle Fatty Acid-Binding Protein

R G Maatman, H T van Moerkerk, I M Nooren, E J van Zoelen, J H Veerkamp

Biochim Biophys Acta. 1994 Aug 25;1214(1):1-10.

PMID: 8068722

Abstract:

Human liver fatty acid-binding protein (L-FABP) has been efficiently expressed in Escherichia coli. The cDNA encoding human liver FABP was under the control of T7 RNA polymerase promoter in the expression vector pET-3b. Expression required overnight induction with isopropyl beta-D-thiogalactopyranoside in the presence of the bacterial RNA polymerase inhibitor, rifampicin. The protein could be purified by (NH4)2SO4 fractionation, anion-exchange and gel filtration chromatography, and was recognized by anti-(human L-FABP) antiserum. The binding characteristics of delipidated recombinant human L-FABP and muscle FABP (M-FABP) for fatty acids of different chain length and saturation grade, and for various hydrophobic ligands, were determined by radiochemical analysis and also by fluorescence for L-FABP. The apparent binding affinity of the ligands was calculated by using displacement curves of oleic acid and dansylamino-undecanoic acid (DAUDA). L-FABP showed a preference for the binding of long-chain saturated and unsaturated fatty acids up to C24:1, whereas the M-FABP has a preference for unsaturated fatty acids, especially those with 18 C atoms. L-FABP also binds palmitoyl derivatives and many other hydrophobic ligands--however, generally with a lower affinity than fatty acids. M-FABP binds--besides with fatty acids--only with oestradiol and testosterone with high affinity. Fatty acids with fluorescent reporter groups are also more tightly bound by L-FABP. A direct assay and displacement study of oleic acid gave the same Kd value of DAUDA for L-FABP. Fluorescence enhancement and displacement studies indicate that the binding of fluorescent fatty acids is determined by both the fluorescent reporter group and the acyl carbon chain.

Chemicals Related in the Paper:

Catalog Number	Product Name	Structure	CAS Number	Price
IAR42414493	DNA Binding Protein-7 (DBP-7), recombinant			Price

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