细胞色素b_5　Glu44、Glu56的定点突变和蛋白质结构比较

Site-directed Mutagenesis at Glu44 and Clu56 of Cytochrome b_5 and Their Structural Comparison with Wild Type Protein

用寡聚核苷酸定点突变的方法将牛肝细胞色素ｂ５基因（编码亲水结构域８２个残基）上第４４位和５６位谷氨酸的密码子ＧＡＡ变成丙氨酸的密码子ＧＣＴ，获得突变体Ｅ４４Ａ、Ｅ５６Ａ和Ｅ４４／５６Ａ的基因。将它们分别克隆于ｐＵＣ１９上，转化大肠杆菌ＪＭ８３后，突变基因得到成功的表达。将含细胞色素ｂ５及其突变体基因的大肠杆菌发酵，酶法裂解细菌，依次用硫酸铵沉淀法、离子交换柱层析、凝胶柱层析进行分离纯化蛋白。用Ｃｈｏｕ－Ｆａｓｍａｎ法、紫外可见光谱、圆二色谱研究蛋白质，发现定点突变细胞色素ｂ５表面上这２个氨基酸残基对蛋白质的局部二级结构无明显影响。

The codon of Glu44 and Glu56,GAA in the gene of trypsin-solubilized bovine liver microsomal cytochrome b5(82 residues in length)was changed into GCT coding for Ala by oligonucleotide site-directed mutagenesis and three cytochrome b5 mutants of E44A,E56A,and E44/56A were obtained.The mutant genes were ligated into ECORⅠ/Hind Ⅲ-cut pUC19and the resulting plasmid was transformed into JM83.All of them were expressed in E. coli successfully.The bacteria containing wild type or mutant cytochrome b5 were dealt with lysozyme, deoxyribonuclease and ribonuclease,then the proteins were isolated and purified by ammonium sulfate precipitation,ion-exchange chromatography and gel filtration.The UV-visible and circular dichroism spectra of purified proteins were studied. The results show that the mutagenesis at the surface residues does not alter the structure of cytochrome b5 significantly.