高等学校化学学报

高等学校化学学报 ›› 1996, Vol. 17 ›› Issue (11): 1673.

• 论文 • 上一篇    下一篇

细胞色素b5Glu44、Glu56的定点突变和蛋白质结构比较

孙雨龙1, 王韵华1, 周刚1, 黄仲贤1, 谢毅2, 吴小舟2   

  1. 1. 复旦大学化学系, 上海, 200433;
    2. 复旦大学遗传研究所
  • 收稿日期:1995-11-29 出版日期:1996-11-24 发布日期:1996-11-24
  • 通讯作者: 黄仲贤
  • 作者简介:孙雨龙,男,28岁,博士研究生.
  • 基金资助:

    国家自然科学基金

Site-directed Mutagenesis at Glu44 and Clu56 of Cytochrome b5 and Their Structural Comparison with Wild Type Protein

SUN Yu-Long1, WANG Yun-Hua1, ZHOU Gang1, HUANG Zhong-Xian1, XIE Yi2, WU Xiao-Zhou2   

  1. 1. Department of Chemistry, Fudan University, Shanghai, 200433;
    2. Institute of Genetics, Fudan University
  • Received:1995-11-29 Online:1996-11-24 Published:1996-11-24

PDF (PC)

摘要： 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。

关键词: 细胞色素b5, 定点突变, 蛋白质结构

Abstract: The codon of Glu44 and Glu56,GAAin the gene of trypsin-solubilized bovine liver microsomal cytochrome b5(82 residues in length)was changed into GCTcoding for Ala by oligonucleotide site-directed mutagenesis and three cytochrome b5 mutants of E44A,E56A,and E44/56 Awere obtained.The mutant genes were ligated into ECORⅠ/Hind Ⅲ-cut pUC19 and 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.

Key words: Cytochrome b5, Site-directed mutagenesis, Protein structure

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