甲硒氨酸耐热邻苯二酚2,3双加氧酶的高表达和分离纯化  

Expression and Purification of Se-Met Substituted Thermostable Catechol 2,3-Dioxygenase

作　　者：季朝能[1] 姜涛[2,3] 殷长传 陈民勤[2,3] 金詟 张志鸿[2,3] 毛裕民[2,3] 

机构地区：[1]复旦大学遗传学研究所,上海200433 [2]复旦大学生命科学学院 [3]复旦大学遗传工程国家重点实验室

出　　处：《复旦学报（自然科学版）》2000年第3期273-276,共4页Journal of Fudan University：Natural Science

基　　金：国家自然科学基金资助项目 (39770 15 7;39730 15 0 )

摘　　要：甲硒氨酸多波长反常散射法是国际上 90年代发展的蛋白质晶体结构测定的新方法 ,该法得以实施的首要一步是获得硒取代 (硫 )的高纯蛋白质 .通过亚克隆将编码耐热邻苯二酚 2 ,3双加氧酶 (简称TCTD)的基因PHEB克隆至表达载体 pRSET ,得到了高表达质粒pRSET PHEB ,转入Met缺陷的菌株B834DE3.通过诱导表达和分离纯化得到了甲硒氨酸参入的TCTD .经质谱测定发现TCTD的所有 7个甲硫氨酸都被甲硒氨酸所替代 ,这为Se Met多波长反常散射法研究TCTD的晶体结构打下了基础 .Se Met multi wavelength anomalous diffraction is a new method for determining protein crystal structure developed in 1990 s. The prerequisite of this method is to obtain Se Met substituted protein. Gene PHEBwhich encodes thermostable catechol 2, 3 dixogenase(TCTD)was subcloned into vector pRSET, and the high expression plasmid pRSET PHEB was constructed. Then pRSET PHEB was transformed into Met defected host B834DE3. TCTD was obtained through induced expression and purification. All seven Mets contained TCTD were found to be substituted with Se Met by mass spectrum, which provided the possibility to determine the crystal structure of TCTD by Se Met multi wavelength anomalous diffraction.

关 键 词：甲硒氨酸多波长反常散射法 表达 纯化 TCTD 

分 类 号：Q550.3[生物学—生物化学]