蛋白质内含子介导的蛋白纯化系统在变性条件下的应用  

Application of intein mediated protein with an affinity tag system(IMPACT) under denature condition

作　　者：汤传龙[1] 施长华[1] 林瑛[1] 孟清[1] 

机构地区：[1]东华大学生物科学与技术研究所,上海201620

出　　处：《生物学杂志》2012年第5期68-71,共4页Journal of Biology

基　　金：国家高技术发展计划(863计划;NO.2006AA03Z451);上海市重点项目(NO.10JC1400300)资助

摘　　要：由蛋白质内含子介导的亲和蛋白质纯化系统(IMPACT)已得到广泛应用,通过其纯化得到的目的蛋白不含蛋白纯化标签以及多余的氨基酸残基,且操作简单成本低廉。这些优点使得其相对于其他蛋白纯化系统有着无与伦比的优势。但是现有报道都局限于非变性条件下使用,这往往会限制其在一些包涵体蛋白变性条件下使用。以一已知表达形成包涵体形式的丝素蛋白为例,研究IMPACT系统在时变性条件下使用变性剂浓度、温度和诱导断裂还原剂浓度。实验表明,在4 M尿素,100 mM DTT室温作用下蛋白质内含子会获得最大断裂效率(80%)。柱上在线断裂实验表明,其最终蛋白得率超过65%。Intein Mediated Protein with An Affinity Tag （IMPACT） system has been widely used in the past years. The purified proteins from this system are without purification tags, additional amino acids, and this system is low cost and easy operation. Although there were many reports of successful application of IMPACT system, nearly all of them were used under nature condition. In this re- port, IMPACT system was used under denatured condition. The concentration of denaturing reagent, thio-reagent and temperature were optimized. The results showed that, the IMPACT system would get the highest cleavage efficiency in the buffer containing 4 M urea, 100 mM DTT at room temperature. The protein recovery is more than 65% on the column.

关 键 词：蛋白质内含子 断裂 变性条件 条件优化 

分 类 号：Q81[生物学—生物工程]