5-Lipoxygenase (5-LO), the key enzyme in leukotriene biosynthesis, is built of a catalytic C-terminal domain and a regulatory N-terminal C2-like domain. The C2-like domain is the target of many regulatory factors or proteins including Ca2+, phospholipids, glycerides, coactosin-like protein and presumably other components that modulate the catalytic activity of 5-LO by acting at this domain, but the detailed underlying molecular mechanisms of these interactions are still unclear. In order to obtain the 5-LO C2-like domain as purified protein in good yields for further mechanistic studies and structure elucidation, a novel expression and purification approach has been applied. A plasmid was constructed expressing a fusion protein of maltose-binding protein (MBP) and the regulatory C2-like domain of 5-LO (AS 1-128), separated by a tobacco etch virus (TEV) protease-cleavage site. The fusion protein MBP-5LO1-128 could be essentially expressed as a soluble protein in Escherichia coli and was efficiently purified by amylose affinity chromatography. By means of this procedure, approximately 80 mg purified fusion protein out of I L E. coli culture were obtained. Digestion with TEV protease yielded the C2-like domain that was further purified using hydrophobic interaction chromatography. Alternatively, the uncleaved fusion protein MBP-5LO1-128 may be suitable to immobilize the C2-like domain on an amylose resin for co-factor interaction studies. Together, we present a convenient expression and purification strategy of the 5-LO C2-like domain that opens many possibilities for structural determination and mechanistic studies, aiming to reveal the precise role and function of this regulatory domain. (C) 2008 Elsevier Inc. All rights reserved.