Direct expression in Escherichia coli and characterization of bovine adrenodoxins with modified amino-terminal regions

• Published in: FEBS letters 1992-04, Vol.300 (3), p.208-212
• Main Authors: Sagara, Yasuhiro, Hara, Takayuki, Ariyasu, Yasuyuki, Ando, Fumiko, Tokunaga, Naomi, Horiuchi, Tadao
• Format: Article
• Language: English
• Subjects: AdR; Adrenodoxin; Adrenodoxin - chemistry; Adrenodoxin - genetics; Adrenodoxin - isolation & purification; Adrenodoxin Reductase; Amino Acid Sequence; Analytical, structural and metabolic biochemistry; Animals; Base Sequence; biochemical characteristics; Biological and medical sciences; Catalysis; Cattle; cDNA; Cloning, Molecular; Cytochrome c; cytochrome P-150 catalyzing steroid 11β-hydroxylation; cytochrome P-450 catalyzing, cholesterol side chain cleavage reaction; Cytochrome P-450 SCC; Cytochrome P-450SCC; Escherichia coli; Escherichia coli - genetics; expression; Expression of protein; Fundamental and applied biological sciences. Psychology; Genetic Vectors; isopropyl-β-d-thiogalactopyranoside; ITPG; M-Ad; mature form of bovine adrenocortical adrenodoxin; Metalloproteins; Molecular Sequence Data; NADPH-adrenodoxin reductase; Other metalloproteins; P-45011β; P-450SCC; Proteins; Recombinant Proteins - chemistry; Recombinant Proteins - isolation & purification; SDS-PAGE; sodium dodecyl sulfate/polyacrylamide gel electrophoresis; Structure-Activity Relationship
• ISSN: 0014-5793; 1873-3468
• DOI: 10.1016/0014-5793(92)80847-A

Four forms of bovine adrenodoxin with modified amino-termini obtained by direct expression of cDNAs in Escherichia coli are Ad(Met 1), Ad(Met −1), Ad(Met −12), and Ad(Met 6). The shoulder numbers represent this site of translation initiator Met at the amino-termini. The adrenodoxins, except for Ad(Met −1), were purified from the cell lysate and the ratios of A 414-to- A 276 of the purified proteins were over 0.92. NADPH-cytochrome c reductase activities of the three forms of adrenodoxin in the presence of adrenodoxin reductase were the same as that of purified bovine adrenocortical adrenodoxin. However, as cytochrome P-450 SCC reduction catalyzed by Ad(Met 0) was about 60% or that by Ad(Met 1), the contribution of the amino-terminal region for the electron transfer or binding to cytochrome P-450 SCC would need to be considered.