Synthesis, bacterial expression, and mutagenesis of the gene coding for mammalian cytochrome b5.
Synthesis, bacterial expression, and mutagenesis of the gene coding for mammalian cytochrome b5.
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哺乳动物细胞色素 b5 编码基因的合成、细菌表达和诱变。
DOI：
10.1073/pnas.83.24.9443
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发表时间：
1986
影响因子：
11.1
通讯作者：
Sligar,SG
中科院分区：
文献类型：
--
作者：
BeckvonBodman,S;Schuler,MA;Jollie,DR;Sligar,SG
我们已经合成了一个编码大鼠肝细胞色素b5的基因。5‘侧翼区是为了在大肠杆菌中高效表达该基因而设计的，它含有一个最适的核糖体结合位点和间隔区。构建并表达了一种类似于酶处理的微粒体蛋白的可溶性形式，以及带有疏水膜锚的完整细胞色素。携带可溶性蛋白基因的转化子产生的细胞色素b5达到细胞总蛋白的8%。完整的细胞色素的表达程度较低，大部分蛋白质存在于细胞膜部分。这代表了哺乳动物金属蛋白基因的完全合成和细菌表达。细胞色素b5通常是一种以组氨酸-39和组氨酸-63为轴向配体的六配位低自旋血红素蛋白。我们通过盒式诱变，利用合成基因中的特定限制酶位点，用蛋氨酸残基取代了组氨酸-63。光学和电子自旋共振光谱表明，合成的蛋白质以组氨酸-39为唯一轴向配体，在铁静息状态下是五配位的高自旋。高产量产生突变细胞色素b5的能力是理解血红素蛋白折叠、蛋白质-蛋白质识别和结合以及生物电子传递过程的关键一步。
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.