Immunocytochemical localization of heterologously expressed adrenodoxin and its electron acceptor cytochrome P45011B1 in Escherichia coli.
Adrenal steroid hydroxylase P45011B1 and its electron donor adrenodoxin were localized in the cortex of bovine adrenals by immunogold-silver staining. In order to test recently developed heterologous expression systems for both enzymes to enable structure-function studies, immunocytochemical marker methods were applied. Adrenodoxin, the ferredoxin of the adrenal gland, was successfully expressed and for the first time localized in Escherichia coli. By use of ultrathin cryosections and the protein A-gold technique, adrenodoxin was detectable in large amounts in the cytoplasm of the bacterial cells, and, following the insertion of the outer membrane protein A leader sequence of E. coli, also in the periplasmic space. A fusion protein between mature adrenodoxin and human P45011B1 was constructed and clearly localized in E. coli by antibodies against both proteins.
Erdmann
B
B
Max Delbrück Center for Molecular Medicine, Berlin-Buch/Germany.
Henning
M
M
Uhlmann
H
H
Beckert
V
V
Grunau
C
C
Bernhardt
R
R
D000302:Adrenal Cortex / Q000737:chemistry; D000325:Adrenodoxin / Q000032:analysis*; D000818:Animals; D001426:Bacterial Proteins / Q000032:analysis*; D015153:Blotting, Western; D002417:Cattle; D019362:Cytochrome P-450 CYP2B1; D003577:Cytochrome P-450 Enzyme System / Q000032:analysis*; D004579:Electron Transport; D004926:Escherichia coli / Q000201:enzymology*; D005629:Frozen Sections; D007150:Immunohistochemistry; D010088:Oxidoreductases / Q000032:analysis*; D011993:Recombinant Fusion Proteins / Q000032:analysis*
D001426:Bacterial Proteins; D011993:Recombinant Fusion Proteins; D000325:Adrenodoxin; D003577:Cytochrome P-450 Enzyme System; D010088:Oxidoreductases; D019362:Cytochrome P-450 CYP2B1