Expression and characterization of human mitochondrial ferredoxin reductase in Escherichia coli.
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, p delta blue; protein expression under control of the bacteriophage lambda pL promoter was then induced in Escherichia 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 apparent Km of the expressed protein for NADPH was determined to be 0.7 +/- 0.1 microM and the apparent Km for human ferredoxin was found to be 106 +/- 8 nM. While yields of active enzyme were relatively low (approximately 0.1 mg/liter of culture), the production of Fd-reductase in E. coli will allow structural and mechanistic studies of the enzyme and its interactions with ferredoxin.
Brandt
M E
ME
Department of Physiology and Biophysics, University of California, Irvine 92717.
Vickery
L E
LE
D001483:Base Sequence; D002942:Circular Dichroism; D003001:Cloning, Molecular; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000235:genetics*; D005287:Ferredoxin-NADP Reductase / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D005822:Genetic Vectors; D006801:Humans; D007700:Kinetics; D008969:Molecular Sequence Data; D008970:Molecular Weight; D010957:Plasmids; D011487:Protein Conformation; D011994:Recombinant Proteins / Q000302:isolation & purification / Q000378:metabolism; D015183:Restriction Mapping
D011994:Recombinant Proteins; D005287:Ferredoxin-NADP Reductase