Soltis D A, McMahon G, Caplan S L, Dudas D A, Chamberlin H A, Vattay A, Dottavio D, Rucker M L, Engstrom R G, Cornell-Kennon S A
Department of Atherosclerosis and Vascular Biology, Sandoz Pharmaceuticals Corporation, East Hanover, New Jersey 07936.
Arch Biochem Biophys. 1995 Feb 1;316(2):713-23. doi: 10.1006/abbi.1995.1095.
We have cloned and utilized a cDNA corresponding to the human squalene synthase gene to generate active enzyme from yeast and baculoviral expression systems. Expression of human squalene synthase in yeast resulted in production of active enzyme in cellular lysates. The presence of the active human enzyme, however, was insufficient to rescue growth of spores defective in yeast squalene synthase function, suggesting that structural differences in the yeast and human enzymes may affect localization or folding of the protein. Expression of the human enzyme in Sf-9 insect cells after infection with recombinant baculovirus encoding the human squalene synthase gene resulted in detection of substantial enzymatic activity in cell lysate preparations. Following extraction from the Sf-9 cells, the human enzyme was purified to near homogeneity utilizing a series of ion-exchange chromatography steps with an overall yield of purified protein of approximately 5 mg per liter of Sf-9 cell culture. The purified enzyme was characterized through steady-state kinetic and physical measurements and the kinetic constants are consistent with values observed for other squalene synthases. Zaragozic acid C was found to be a competitive inhibitor with respect to farnesyl pyrophosphate and has a Kis value of 250 pM (@ [NADPH] = 5 mM). Inhibition experiments with zaragozic acid C at low (approximately 0.5 x Km) and high (approximately 10 x Km) concentrations of NADPH indicated that the inhibitor does not bind in the enzyme's NADPH binding domain. These studies demonstrate that the human enzyme can be prepared from baculovirus-infected Sf-9 cells in a catalytically active configuration and in sufficient quantities to allow for further biochemical, kinetic, and structural characterization.
我们克隆并利用了与人类鲨烯合酶基因对应的cDNA，以从酵母和杆状病毒表达系统中产生活性酶。人类鲨烯合酶在酵母中的表达导致细胞裂解物中产生活性酶。然而，活性人类酶的存在不足以挽救酵母鲨烯合酶功能缺陷的孢子的生长，这表明酵母和人类酶的结构差异可能会影响蛋白质的定位或折叠。在用编码人类鲨烯合酶基因的重组杆状病毒感染后，在Sf-9昆虫细胞中表达人类酶，结果在细胞裂解物制剂中检测到大量酶活性。从Sf-9细胞中提取后，利用一系列离子交换色谱步骤将人类酶纯化至接近均一，每升Sf-9细胞培养物中纯化蛋白质的总产率约为5mg。通过稳态动力学和物理测量对纯化的酶进行了表征，其动力学常数与其他鲨烯合酶观察到的值一致。发现扎拉戈昔酸C是法呢基焦磷酸的竞争性抑制剂，其Kis值为250 pM（@[NADPH]=5 mM）。在低（约0.5×Km）和高（约10×Km）浓度的NADPH下用扎拉戈昔酸C进行的抑制实验表明，该抑制剂不结合在酶的NADPH结合结构域中。这些研究表明，人类酶可以从杆状病毒感染的Sf-9细胞中以催化活性形式制备，并且数量足以进行进一步的生化、动力学和结构表征。
