Synthesis, bacterial expression, and mutagenesis of the gene coding for mammalian cytochrome b5.
- 1 December 1986
- journal article
- Published by Proceedings of the National Academy of Sciences in Proceedings of the National Academy of Sciences
- Vol. 83 (24), 9443-9447
- https://doi.org/10.1073/pnas.83.24.9443
Abstract
We have totally synthesized a gene that codes for rat hepatic cytochrome b5. The 5' flanking region was designed for efficient expression of this gene in Escherichia coli by incorporating an optimum ribosome binding site and spacer region. Both a soluble form, analogous to the protease-treated microsomal protein, as well as the complete cytochrome with hydrophobic membrane anchor, was constructed and expressed. Transformants with the gene for the soluble protein overproduce authentic cytochrome b5 to a level of 8% of the total cell protein. The complete cytochrome is expressed to a lesser extent with most of the protein found in the cell membrane fraction. This represents complete synthesis and bacterial expression of a mammalian metalloprotein gene. Cytochrome b5 is normally a six-coordinate low spin heme protein with histidine-39 and histidine-63 as axial ligands. We have replaced histidine-63 with a methionine residue by cassette mutagenesis, utilizing specific restriction enzyme sites engineered into the synthetic gene. The resultant protein has histidine-39 as sole axial ligand and is five-coordinate high spin in the ferric resting state, as indicated by optical and electron spin resonance spectroscopy. The ability to generate mutant cytochrome b5 in high yield is a crucial step in understanding heme protein folding, protein-protein recognition and binding, and biological electron transfer processes.Keywords
This publication has 16 references indexed in Scilit:
- Nucleotide sequence of the Pseudomonas putida cytochrome P-450cam gene and its expression in Escherichia coli.Journal of Biological Chemistry, 1986
- Chemical characterization of protein-protein interactions between cytochrome P-450 and cytochrome b5.Journal of Biological Chemistry, 1985
- Generation of β-globin by sequence-specific proteolysis of a hybrid protein produced in Escherichia coliNature, 1984
- Reconstitution of Hepatic Microsomal Stearoyl-Coenzyme A Desaturase System from Solubilized Components*The Journal of Biochemistry, 1972
- The Structure of Cytochrome b5 at 2.0 A ResolutionCold Spring Harbor Symposia on Quantitative Biology, 1972
- A Form of Cytochrome b 5 That Contains an Additional Hydrophobic Sequence of 40 Amino Acid ResiduesProceedings of the National Academy of Sciences, 1971
- Evidence for the participation of cytochrome b5 in hepatic microsomal mixed-function oxidation reactionsArchives of Biochemistry and Biophysics, 1971
- Microsomal electron transport reactionsArchives of Biochemistry and Biophysics, 1971
- Catalysis of Methaemoglobin Reduction by Erythrocyte Cytochrome b5 and Cytochrome b5 ReductaseNature New Biology, 1971
- Stimulation by Phenols of the Reoxidation Microsomal Bound Cytochrome b5 and Its Implication to Fatty Acid DesaturationThe Journal of Biochemistry, 1971