RGD Reference Report - High-level expression in Escherichia coli of the soluble, catalytic domain of rat hepatic cytochrome b5 reductase. - Rat Genome Database

Send us a Message
Submit Data |  Help |  Video Tutorials |  News |  Publications |  Download |  REST API |  Citing RGD |  Contact   
Search RGD Grant Resources Citing RGD About Us Contact Us
Genes Variants Community Projects QTLs Strains Markers Genome Information Ontologies Cell Lines References Download Submit Data
OntoMate (Literature Search) JBrowse (Genome Browser) Synteny Browser (VCMap) Variant Visualizer Multi-Ontology Enrichment (MOET) Gene-Ortholog Location Finder (GOLF) InterViewer (Protein-Protein Interactions) PhenoMiner (Quatitative Phenotypes) Gene Annotator OLGA (Gene List Generator) AllianceMine GViewer (Genome Viewer)
Aging & Age-Related Disease Cancer & Neoplastic Disease Cardiovascular Disease Coronavirus Disease Developmental Disease Diabetes Hematologic Disease Immune & Inflammatory Disease Infectious Disease Liver Disease Neurological Disease Obesity & Metabolic Syndrome Renal Disease Respiratory Disease Sensory Organ Disease
Find Models   new Genetic Models Autism Models Rat PhenoMiner (Quantitative Phenotypes) Chinchilla PhenoMiner Expected Ranges (Quantitative Phenotype) PhenoMiner Term Comparison Hybrid Rat Diversity Panel Phenotypes Phenotypes in Other Animal Models Animal Husbandry Strain Medical Records Phylogenetics Strain Availability Calendar Rats 101 Submissions Photo Archive
Pathways
Rat Community Forum Directory of Rat Laboratories Video Tutorials News RGD Publications RGD Presentations Archive Nomenclature Guidelines Resource Links Laboratory Resources Employment Resources

High-level expression in Escherichia coli of the soluble, catalytic domain of rat hepatic cytochrome b5 reductase.

Authors: Barber, M J  Quinn, G B 
Citation: Barber MJ and Quinn GB, Protein Expr Purif. 1996 Aug;8(1):41-7. doi: 10.1006/prep.1996.0072.
RGD ID: 153298951
Pubmed: PMID:8812833   (View Abstract at PubMed)
DOI: DOI:10.1006/prep.1996.0072   (Journal Full-text)

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.



Gene Ontology Annotations    Click to see Annotation Detail View

Molecular Function
Object SymbolSpeciesTermQualifierEvidenceWithNotesSourceOriginal Reference(s)
Cyb5r3Ratcytochrome-b5 reductase activity, acting on NAD(P)H enablesIDA PMID:8812833UniProt 
Cyb5r3RatFAD binding enablesIDA PMID:8812833UniProt 

Objects Annotated

Genes (Rattus norvegicus)
Cyb5r3  (cytochrome b5 reductase 3)


Additional Information