Direct expression of adrenodoxin reductase in Escherichia coli and the functional characterization.
A plasmid for direct expression in Escherichia coli of the mature form bovine adrenodoxin reductase was constructed from the full-size cDNA for the enzyme [Y. Sagara, Y. Takata, T. Miyata, T. Hara, and T. Horiuchi, J. Biochem. (Tokyo), 102, 1333 (1987)] and an expression vector pCWori+. The recombinant adrenodoxin reductase was purified from the transformed E. coli cell lysates using adrenodoxin-Sepharose affinity chromatography [T. Sugiyama and T. Yamano, FEBS Lett., 52, 145 (1975)] with a yield of 2.5 mg/l of culture. The purified recombinant enzyme showed a single band on polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate and migration was identical with that of the authentic enzyme purified from bovine adrenal cortex mitochondria. The recombinant enzyme had Ser at its amino-terminus and the sequence of the amino terminal 9 residues was identical with that of the authentic bovine enzyme. The absorption spectrum of the recombinant enzyme showed peaks at 270, 376, and 450 nm and shoulders at 425 and 475 nm. Flavin content of the recombinant enzyme was 0.8 mol FAD/mol. The apparent Km value for bovine adrenodoxin in NADPH-cytochrome c reductase activity using a reconstitution system was 16 nM, a value comparable with that of the authentic bovine enzyme (17 nM). The cholesterol side chain cleavage activity with a reconstitution system was about 75% of that obtained when the authentic enzyme was used.
Sagara
Y
Y
Department of Microbiology, Faculty of Pharmaceutical Sciences, Kyushu University, Fukuoka, Japan.
Wada
A
A
Takata
Y
Y
Waterman
M R
MR
Sekimizu
K
K
Horiuchi
T
T
D000302:Adrenal Cortex / Q000201:enzymology; D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D002417:Cattle; D002846:Chromatography, Affinity; D003470:Culture Media; D018076:DNA, Complementary / Q000737:chemistry / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000201:enzymology* / Q000235:genetics; D005287:Ferredoxin-NADP Reductase / Q000096:biosynthesis* / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D005415:Flavins / Q000032:analysis; D007700:Kinetics; D008969:Molecular Sequence Data; D010957:Plasmids; D011994:Recombinant Proteins / Q000096:biosynthesis; D013056:Spectrophotometry, Ultraviolet
D003470:Culture Media; D018076:DNA, Complementary; D005415:Flavins; D011994:Recombinant Proteins; D005287:Ferredoxin-NADP Reductase