RRC ID 71450
Author Sagara Y, Aramaki H.
Title Overproduction in Escherichia coli and characterization of the precise mature form of rat adrenodoxin.
Journal Biol Pharm Bull
Abstract The mature form of rat adrenodoxin (Ad) was purified from a heterologous direct expression system in Escherichia coli with a yield of 56 mg/l culture. The purified Ad showed a A414/A280 ratio of 0.91 and the sequence of 10 amino terminal residues was identical with that of authentic rat Ad. By Time of Flight/Mass spectrometry, the molecular mass of purified Ad was identical to that calculated from the cDNA sequence and the carboxy terminal residue was estimated to be Ser, which was also as expected from the cDNA. These results indicate that the purified recombinant Ad is a precise mature form. In measurements of NADPH-cytochrome c reductase activity reconstituted with bovine adrenodoxin reductase (AdR), the apparent Km value for rat Ad was 46.9+/-2.5 nM, indicating a somewhat lower affinity for rat Ad to bovine AdR than for bovine Ad. On the other hand, the spectral Kd value for rat Ad to bovine cytochrome P-450scc was 0.46+/-0.05 microM, a value which was almost identical with that of the bovine counterpart.
Volume 21(10)
Pages 1106-9
Published 1998-10-1
DOI 10.1248/bpb.21.1106
PMID 9821820
MeSH Adrenodoxin / biosynthesis* Adrenodoxin / genetics Adrenodoxin / isolation & purification Animals Cattle DNA / genetics DNA / metabolism Electrophoresis, Polyacrylamide Gel Escherichia coli / genetics Escherichia coli / metabolism* Kinetics NADPH-Ferrihemoprotein Reductase / metabolism Plasmids Rats Recombinant Proteins / biosynthesis Recombinant Proteins / genetics Recombinant Proteins / isolation & purification Sodium Dodecyl Sulfate
IF 1.863
DNA material pRA3813 (RDB19562)