High-level expression in Escherichia coli of the soluble, catalytic domain of rat hepatic cytochrome b5 reductase.
A T7 expression system has been produced for the high-level production of the soluble, catalytic domain of rat hepatic cytochrome b5 reductase in Escherichia coli. The recombinant protein was purified to homogeneity using affinity chromatography on 5'-ADP agarose and gel exclusion chromatography and exhibited a molecular mass of approximately 30 kDa by polyacrylamide gel electrophoresis and a molecular mass of 30,588 by mass spectrometry. Direct sequencing of the initial 12 residues of the amino-terminus of the purified domain yielded the sequence MITLENPDIKYP, identical to that predicted from the DNA sequence. The domain incorporated a full complement of FAD with a visible absorption spectrum typical of a flavoprotein exhibiting maxima at 389 and 461 nm and a distinct shoulder at 485 nm. Addition of NADH to the protein resulted in an extensive bleaching of the visible spectrum. The recombinant domain retained both NADH:ferricyanide and NADH:cytochrome b5 reductase activities with Vmax of 48 and 26 micromol NADH consumed/min/nmol FAD, respectively, and Km of 6, 7, and 11 microM for NADH, ferricyanide, and cytochrome b5. Comparison of the activities obtained using NADH and NADPH indicated a substantial preference for NADH as the reducing substrate. The results indicate that the recombinant protein retains the physical and catalytic properties of the native protein and represents an excellent system for probing the role of specific amino acid residues using site-directed mutagenesis.
Barber
M J
MJ
Department of Biochemistry and Molecular Biology, University of South Florida, College of Medicine, Tampa, Florida, 33612, USA.
Quinn
G B
GB
D000818:Animals; D002850:Chromatography, Gel; D002851:Chromatography, High Pressure Liquid; D003579:Cytochrome Reductases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D042966:Cytochrome-B(5) Reductase; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000235:genetics; D005182:Flavin-Adenine Dinucleotide / Q000378:metabolism; D015870:Gene Expression / Q000235:genetics; D007700:Kinetics; D008099:Liver / Q000201:enzymology; D013058:Mass Spectrometry; D009243:NAD / Q000378:metabolism; D009249:NADP / Q000378:metabolism; D010957:Plasmids / Q000235:genetics; D016133:Polymerase Chain Reaction; D051381:Rats; D011994:Recombinant Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D013053:Spectrophotometry
D011994:Recombinant Proteins; D009243:NAD; D005182:Flavin-Adenine Dinucleotide; D009249:NADP; D003579:Cytochrome Reductases; D042966:Cytochrome-B(5) Reductase