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.