期刊
ANALYTICAL BIOCHEMISTRY
卷 317, 期 2, 页码 180-185出版社
ACADEMIC PRESS INC ELSEVIER SCIENCE
DOI: 10.1016/S0003-2697(03)00138-6
关键词
farnesyl diphosphate; isoprenoid; isoprenoid diphosphate
A sensitive method was developed for measuring farnesyl diphosphate (FPP) accumulation in a mutant strain of Saccharomyces cereviside. The strain was blocked at squalene synthase (ERG9 gene) in the isoprenoid pathway and had the catalytic domain of the 3-hydroxy-3-methylglutaryl coenzyme A reductase gene integrated into the chromosome. It required ergosterol for growth and produced E, E-farnesol. The method was based on the isolation of FPP using the anion exchanger Macro Prep High Q and conversion of FPP to E, E-farnesol with alkaline phosphatase. Farnesol was measured using gas chromatography-mass spectrometry. Background farnesol in the cell-free extract was also retained by the anion exchanger, but was removed with repeated washing with methanol. Both 1 M NaCl and 40% (v/v) methanol were required in the elution buffer to effectively elute FPP. The preparation of cell-free extract in Bis-Tris propane/HCI, pH 7, buffer containing 0.025% (w/v) Triton X-100 and 15 mM MgCl2 provided optimum conditions for the stabilization of FPP. (C) 2003 Elsevier Science (USA). All rights reserved.
作者
我是这篇论文的作者
点击您的名字以认领此论文并将其添加到您的个人资料中。
推荐
暂无数据