检测Sortase A酶催化蛋白质连接效率的新型报道系统

A novel reporter system monitoring Sortase A catalyzed protein ligation efficiency

摘要:

利用分子进化技术对Sortase A酶的催化活性进行定向改造已经成为当前的研究热点和重点，为了高效筛选特定催化活性的Sortase A酶突变体，需要建立一种能够快速准确鉴定Sortase A酶活性的方法。为此，设计了由GFP-LPETG蛋白和GGGYK-Biotin组成的新型报道底物系统。利用重组基因工程技术，成功制备GFP-LPETG蛋白，野生型Sortase A酶及近期报道的一个高活性突变型Sortase A酶。以GFP-LPETG及GGGYK-Biotin为报道底物建立连接体系，结果显示，连接反应动力学可直接通过SDS-PAGE凝胶电泳法精确测定。用此连接体系比较野生型与突变型Sortase A酶催化的连接反应效率，证实高活性突变酶具有野生酶无法比拟的高催化活性。此报道系统简单快速灵敏，可应用于系统的筛选，为后续进一步定向优化Sortase A酶奠定了基础。

Abstract:

Efforts on directed evolution of sortase A to optimize its catalytic properties have been undertaken and shown the promise. To facilitate screening of sortase A mutants with expected catalytic properties, a novel ligation efficiency monitoring system, including reporter substrates GFP-LPETG and GGGYK-Biotin, was developed. GFP-LPETG, wild type sortase A, and a recently reported high activity sortase A mutant were prepared recombinantly from Escherichia coli BL21 (DE3). Taking advantage of the newly designed reporter system, the ligation efficiency catalyzed by wild type and mutant form of sortase A could be sensitively monitored via SDS-PAGE directly. Consistent with previous report, the mutant sortase A displayed much higher catalytic activity compared to wild type enzyme, indicating the new reporter system is easily and fast handled and sensitive. The application of this reporter system into systemic screening will facilitate future directed optimization of sortase A.