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Variant cDNA sequences of human ATP:citrate lyase: cloning, expression, and purification from baculovirus-infected
K A Lord1, X M Wang, S J Simmons
1Department of Molecular and Cellular Biology, Sterling Winthrop Pharmaceutical Research Division, Collegeville, Pennsylvania 19426, USA.
Abstract:
ATP:citrate lyase (ACL) is a major generator of cytosolic acetyl-coenzymeA, which is required for both fatty acid and cholesterol biosynthesis. The human ACL (hACL) cDNA was cloned by RT-PCR, and our results indicate the existence of previously unknown sequence variations in hACL. Expression of the hACL cDNA in Spodoptera frugiperda 9 insect cells resulted in the production of high levels of soluble, active enzyme. The recombinant protein (re-hACL) was purified to homogeneity from the soluble lysate of infected cells and was observed to exist as a tetramer by gel filtration chromatography. Kinetic analyses indicated that the re-hACL and rat ACL have very similar enzymological properties. The facile preparation of milligram quantities of purified, active re-hACL affords the opportunity to characterize the enzyme for structure-based design of hypolipidemic drugs, and to further examine the functional significance of the sequence variations.