EXPRESSION AND CHARACTERIZATION OF HUMAN MITOCHONDRIAL FERREDOXIN REDUCTASE IN ESCHERICHIA-COLI
EXPRESSION AND CHARACTERIZATION OF HUMAN MITOCHONDRIAL FERREDOXIN REDUCTASE IN ESCHERICHIA-COLI
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DOI：
10.1016/0003-9861(92)90749-m
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发表时间：
1992-05-01
影响因子：
3.9
通讯作者：
VICKERY, LE
中科院分区：
文献类型：
--
作者：
BRANDT, ME;VICKERY, LE
Ferredoxin reductase (Fd-reductase) supplies reducing equivalents obtained from NADPH to mitochondrial cytochrome P450 enzymes via the small iron-sulfur protein ferredoxin. Two cDNAs (differing by the presence or absence of an 18-bp insert in the coding region) for the human Fd-reductase were subcloned into a newly constructed general purpose expression vector, pΔblue; protein expression under control of the bacteriophage λpLpromoter was then induced inEscherichia coli. Western blot analysis of subcellular fractions indicated that Fd-reductase protein expressed from both plasmids was present in both inclusion bodies and soluble fractions. However, only the form lacking the insert exhibited Fd-reductase activity. The active material was purified and was found to have electrophoretic, chromatographic, optical, and circular dichroism properties comparable to the bovine homologue. The apparentKmof the expressed protein for NADPH was determined to be 0.7 ± 0.1 μmand the apparentKmfor human ferredoxin was found to be 106 ± 8 nm. While yields of active enzyme were relatively low (~0.1 mg/liter of culture), the production of Fd-reductase inE. coliwill allow structural and mechanistic studies of the enzyme and its interactions with ferredoxin.
