致死性甲基化酶M.TneDI在大肠杆菌BL21(DE3)中的过表达。

Overexpression of a lethal methylase, M.TneDI, in E. coli BL21(DE3).

作者信息

Xu Hui, Han Dongmei, Xu Zhaohui

机构信息

Department of Biological Sciences, Bowling Green State University, Bowling Green, OH, 43403, USA,

出版信息

Biotechnol Lett. 2014 Sep;36(9):1853-9. doi: 10.1007/s10529-014-1552-z. Epub 2014 May 28.

DOI:10.1007/s10529-014-1552-z

PMID:24867319

Abstract

A pET-based vector pDH21 expressing the methylase, M.TneDI (recognizing CGCG) from Thermotoga was constructed, and transformed into E. coli BL21(DE3). Despite E. coli BL21(DE3) being McrBC positive, 30 transformants were isolated, which were suspected to be McrBC(-) mutants. The overexpression of M.TneDI was verified by SDS-PAGE analysis. Compared to the previously constructed pJC340 vector, a pACYC184 derivative expressing M.TneDI from a tet promotor, the newly constructed pDH21 vector improved the expression of the methylase about fourfold, allowing complete protection of DNA substrates. This study not only demonstrates a practical approach to overexpressing potential lethal proteins in E. coli but also delivers a production strain of M.TneDI that may be useful in various in vitro methylation applications.

摘要

构建了一种基于pET的载体pDH21，其表达来自嗜热栖热菌的甲基化酶M.TneDI（识别CGCG），并将其转化到大肠杆菌BL21(DE3)中。尽管大肠杆菌BL21(DE3)是McrBC阳性，但仍分离出30个转化体，怀疑它们是McrBC(-)突变体。通过SDS-PAGE分析验证了M.TneDI的过表达。与先前构建的从tet启动子表达M.TneDI的pACYC184衍生物pJC340载体相比，新构建的pDH21载体使甲基化酶的表达提高了约四倍，从而能够完全保护DNA底物。本研究不仅展示了在大肠杆菌中过表达潜在致死蛋白的实用方法，还提供了一种M.TneDI生产菌株，可用于各种体外甲基化应用。