不依赖天然外显子的蛋白质内含子定向进化筛选系统

蛋白质剪接技术为在蛋白质水平上直接对蛋白质进行修饰和加工提供了一种全新的解决方案,因而在蛋白质工程及相关领域具有非常广阔的应用前景。现阶段,大部分天然的蛋白质内含子在异源蛋白质中剪接活性非常低,极大限制了蛋白质内含子的开发和应用。为了开发一个可以同时对蛋白质内含子通用性和剪接活性进行筛选的系统,利用Bsa I限制性内切酶识别位点和切割不重合的特性,将Ter ThyX内含子(不含外显子序列)插入到卡那霉素抗性蛋白基因的多个位点。并且摒弃了以往需要结合天然外显子以实现剪接的方法,可以同时对蛋白质内含子的剪接活性和通用性进行筛选。Western blot结果和卡那霉素平板生长结果表明,通过卡那霉素筛选系统可以精确的将蛋白质内含子剪接反应与卡那霉素抗性结合起来,仅从卡那霉素平板上的菌落生长情况即可完成蛋白质内含子剪接活性阳性突变的筛选,是一个快速,稳定的定向进化筛选系统。

Natural Extein Free Screening System for Intein Directed-Evolution

Intein-mediated protein splicing is a brand new solution for protein artificial modification, thus possessing broad application foreground in protein engineering. So far, most inteins present low splicing efficiency in heterogeneous protein which highly limited the intein development and application. In order to develop an high throughput screening system for intein directed-evolution, the Bsa I enzyme was utilized to insert Ter ThyX intein(TX intein) coding sequence into kanamycin resistance protein sequence. Kanamycin screening system can combine the intein-splicing and kanamycin resistance, which is proved by the kanamycin plate assay and Western blot assay. This is an extein free screening system, which is capable of screening the splicing activity and the versatility at the same time. The spliced intein can be screened simply by picking the clone that can survive on the kanamycin plate. The screening system proved to be an efficiency, stable screening system.