ONE-STEP PURIFICATION OF RAT HEART-TYPE FATTY-ACID-BINDING PROTEIN EXPRESSED IN ESCHERICHIA-COLI

Heart-type fatty acid-binding protein (H-FABP) is a member of a family of 14-15 kDa lipid binding proteins which are believed to enhance int racellular transport of lipids by facilitating their cytoplasmic diffu sion. To obtain sufficient amounts of protein for in vitro studies, we expressed rat H-FABP in Escherichia coli and compared its biochemical properties with the protein isolated from rat heart. An effective met hod was developed to purify recombinant rat H-FABP from cell lysates i n a single step using anion-exchange chromatography. This method also proved to be applicable for purifying heterologously expressed human H -FABP. Recombinant rat H-FABP, which made up approximately 25% of the soluble proteins in E. coli, was obtained in a yield of 30-40 mg/l cul ture. Characterization showed that recombinant rat H-FABP was indistin guishable from the protein isolated from rat heart regarding molecular mass and oleic acid binding. Some heterogeneity upon isoelectric focu sing was observed, presumably due to differences in N-terminal process ing of the proteins. In conclusion, a method is presented for efficien t high-yield production of recombinant rat H-FABP.