Synthesis, bacterial expression, and mutagenesis of the gene coding for mammalian cytochrome b5

S. Beck von Bodman, M. A. Schuler, D. R. Jollie, S. G. Sligar

Research output: Contribution to journalArticlepeer-review

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 fractions. 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.

Original languageEnglish (US)
Pages (from-to)9443-9447
Number of pages5
JournalProceedings of the National Academy of Sciences of the United States of America
Volume83
Issue number24
DOIs
StatePublished - 1986

ASJC Scopus subject areas

  • General

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