EXPRESSION OF HUMAN LIVER FATTY-ACID-BINDING PROTEIN IN ESCHERICHIA-COLI AND COMPARATIVE-ANALYSIS OF ITS BINDING CHARACTERISTICS WITH MUSCLE FATTY-ACID-BINDING PROTEIN

Human liver fatty acid-binding protein (L-FABP) has been efficiently e xpressed in Escherichia coli. The cDNA encoding human liver FABP was u nder the control of T7 RNA polymerase promoter in the expression vecto r pET3b. 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)(2)SO4 f ractionation, anion-exchange and gel filtration chromatography, and wa s recognized by anti-(human L-FABP) antiserum. The binding characteris tics of delipidated recombinant human L-FABP and muscle FABP (M-FABP) for fatty acids of different chain length and saturation grade, and fo r various hydrophobic ligands, were determined by radiochemical analys is and also by fluorescence for L-FABP. The apparent binding affinity of the ligands was calculated by using displacement curves of oleic ac id and dansylamino-undecanoic acid (DAUDA). L-FABP showed a preference for the binding of long-chain saturated and unsaturated fatty acids u p to C-24:1, 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 wi th a lower affinity than fatty acids. M-FABP binds - besides with fatt y acids - only with oestradiol and testosterone with high affinity. Fa tty acids with fluorescent reporter groups are also more tightly bound by L-FABP. A direct assay and displacement study of oleic acid gave t he same K-d value of DAUDA for L-FABP. Fluorescence enhancement and di splacement studies indicate that the binding of fluorescent fatty acid s is determined by both the fluorescent reporter group and the acyl ca rbon chain.