CRISPR系统相关蛋白的纯化与活性分析

Purification and activity analysis of proteins related to CRISPR system

目的对CRISPR相关蛋白Cas3、Cas6及Cas9进行表达纯化,并测定其活性。方法在体外构建带有目的基因的质粒,导入大肠杆菌表达系统,通过大肠杆菌的过表达,裂解并提取蛋白,利用His标签,可将目的蛋白纯化出来;通过3种Cas蛋白对不同核酸的切割,分别设计3种核酸底物,再通过聚丙烯酰胺凝胶电泳,最终确定Cas蛋白的活性。结果成功表达并纯化得到Cas3、Cas6及Cas93种蛋白,并根据其蛋白特性,与相应的核酸底物进行孵育,在电泳后通过凝胶成像,可观察到3种蛋白均具有活性。结论表达纯化的Cas3、Cas6及Cas9具有生物学功能,为其功能机制研究奠定了基础。

Objective To expression and purify CRISPR-related proteins: Cas3, Cas6 and Cas9, and determine their activity to lay the foundation for the subsequent screening of their inhibitors. Methods The plasmid carrying the target gene was constructed in vitro, introduced into an E. coli expression system, and the protein was extracted by over-expression and lysis of E. coli, and the target protein was purified by using the His tag;the cleavage of different nucleic acids by the three Cas proteins was performed. Three nucleic acid substrates were designed respectively, and then the activity of Cas protein was finally determined by polyacrylamide gel electrophoresis. Results Three proteins, Cas3, Cas6 and Cas9, were successfully expressed and purified, and incubated with the corresponding nucleic acid substrate according to their protein properties. After electrophoresis, gel imaging showed that all three proteins were active. Conclusion Three active proteins, Cas3, Cas6 and Cas9, were obtained, which provided the basis for the subsequent screening of anti-CRISPR system.