ANALYSIS OF THE LIGAND-BINDING PROPERTIES OF RECOMBINANT BOVINE LIVER-TYPE FATTY-ACID-BINDING PROTEIN

The coding part of the cDNA for bovine liver-type fatty acid binding p rotein (L-FABP) has been amplified by RT-PCR, cloned and used for the construction of an Escherichia coli (E. coli) expression system. The r ecombinant protein made up to 25% of the soluble E. coli proteins and could be isolated by a simple two step protocol combining ion exchange chromatography and gel filtration. Dissociation constants for binding of oleic acid, arachidonic acid, oleoyl-CoA, lysophosphatidic acid an d the peroxisomal proliferator bezafibrate to L-FABP have been determi ned by titration calorimetry. All ligands were bound in a 2:1 stoichio metry, the dissociation constants for the first ligand bound were all in the micro molar range, Oleic acid was bound with the highest affini ty and a K-d of 0.26 mu M. Furthermore, binding of cholesterol to L-FA BP was investigated with the Lipidex assay, a liposome binding assay a nd a fluorescence displacement assay. In none of the assays binding of cholesterol to L-FABP was observed.