Variant cDNA sequences of human ATP:citrate lyase: cloning, expression, and purification from baculovirus-infected insect cells.
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.
Lord
K A
KA
Department of Molecular and Cellular Biology, Sterling Winthrop Pharmaceutical Research Division, Collegeville, Pennsylvania 19426, USA.
Wang
X M
XM
Simmons
S J
SJ
Bruckner
R C
RC
Loscig
J
J
O'Connor
B
B
Bentley
R
R
Smallwood
A
A
Chadwick
C C
CC
Stevis
P E
PE
Ciccarelli
R B
RB
D001275:ATP Citrate (pro-S)-Lyase / Q000096:biosynthesis / Q000235:genetics* / Q000302:isolation & purification*; D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D003001:Cloning, Molecular; D018076:DNA, Complementary / Q000235:genetics*; D015870:Gene Expression; D014644:Genetic Variation; D005822:Genetic Vectors; D006801:Humans; D007700:Kinetics; D008969:Molecular Sequence Data; D017924:Nucleopolyhedroviruses / Q000235:genetics; D016133:Polymerase Chain Reaction; D051381:Rats; D011994:Recombinant Proteins / Q000096:biosynthesis; D013045:Species Specificity; D018411:Spodoptera / Q000166:cytology / Q000821:virology
D018076:DNA, Complementary; D011994:Recombinant Proteins; D001275:ATP Citrate (pro-S)-Lyase