Development of a method for expression and purification of the regulatory C2-like domain of human 5-lipoxygenase.
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 Ca(2+), 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 80mg purified fusion protein out of 1L 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.
Michel
Angela A Y
AA
Institute of Pharmaceutical Chemistry, University of Frankfurt, Max-von-Laue-Str. 9, D-60438 Frankfurt, Germany.
Steinhilber
Dieter
D
Werz
Oliver
O
D001094:Arachidonate 5-Lipoxygenase / Q000096:biosynthesis* / Q000302:isolation & purification; D002851:Chromatography, High Pressure Liquid; D010450:Endopeptidases / Q000378:metabolism; D006801:Humans; D017434:Protein Structure, Tertiary; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000302:isolation & purification
D011993:Recombinant Fusion Proteins; D001094:Arachidonate 5-Lipoxygenase; D010450:Endopeptidases; C099488:TEV protease