EXPRESSION, PURIFICATION, AND CHARACTERIZATION OF THE HUMAN SQUALENE SYNTHASE - USE OF YEAST AND BACULOVIRAL SYSTEMS

We have cloned and utilized a cDNA corresponding to the human squalene synthase gene to generate active enzyme from yeast and baculoviral ex pression systems. Expression of human squalene synthase in yeast resul ted in production of active enzyme in cellular lysates. The presence o f the active human enzyme, however, was insufficient to rescue growth of spores defective in yeast squalene synthase function, suggesting th at structural differences in the yeast and human enzymes may affect lo calization or folding of the protein. Expression of the human enzyme i n Sf-9 insect cells after infection with recombinant baculovirus encod ing the human squalene synthase gene resulted in detection of substant ial enzymatic activity in cell lysate preparations. Following extracti on from the Sf-9 cells, the human enzyme was purified to near homogene ity utilizing a series of ion-exchange chromatography steps with an ov erall yield of purified protein of approximately 5 mg per liter of Sf- 9 cell culture. The purified enzyme was characterized through steady-s tate kinetic and physical measurements and the kinetic constants are c onsistent with values observed for other squalene synthases. Zaragozic acid C was found to be a competitive inhibitor with respect to farnes yl pyrophosphate and has a K-is value of 250 pM (@ [NADPH] = 5 mM). In hibition experiments with zaragozic acid C at low (similar to 0.5 X K- m) and high (similar to 10 X K-m) concentrations of NADPH indicated th at the inhibitor does not bind in the enzyme's NADPH binding domain. T hese studies demonstrate that the human enzyme can be prepared from ba culovirus-infected Sf-9 cells in a catalytically active configuration and in sufficient quantities to allow for further biochemical, kinetic , and structural characterization. (C) 1995 Academic Press, Inc.