VARIANT CDNA SEQUENCES OF HUMAN ATP-CITRATE LYASE - CLONING, EXPRESSION, AND PURIFICATION FROM BACULOVIRUS-INFECTED INSECT CELLS

摘要

ATP:citrate lyase (ACL) is a major generator of cytosolic acetyl-coenzyme A, 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. (C) 1997 Academic Press. [References: 27]