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标题：Synthesis, bacterial expression, and mutagenesis of the gene coding for mammalian cytochrome b5
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作者：S Beck von Bodman ; M A Schuler ; D R Jollie 等
期刊名称：Proceedings of the National Academy of Sciences
印刷版ISSN：0027-8424
电子版ISSN：1091-6490
出版年度：1986
卷号：83
期号：24
页码：9443-9447
DOI：10.1073/pnas.83.24.9443
语种：English
出版社：The National Academy of Sciences of the United States of America
摘要：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.