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

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

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

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.