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. 2011 Nov;15(6):665-72.
doi: 10.1007/s00792-011-0397-9. Epub 2011 Sep 15.

Cloning and characterization of the TneDI restriction: modification system of Thermotoga neapolitana

Zhaohui Xu  1 , Dongmei Han, Jingjing Cao, Uksha Saini
Affiliations

Cloning and characterization of the TneDI restriction: modification system of Thermotoga neapolitana

Zhaohui Xu et al. Extremophiles. 2011 Nov.

Abstract

A putative Type II restriction-modification system of Thermotoga neapolitana, TneDI, was cloned into Escherichia coli XL1-Blue MRF' and characterized. Gene CTN_0339 specifies the endonuclease R.TneDI, while CTN_0340 encodes the cognate DNA methyltransferase M.TneDI. Both enzymes were purified simply by heating the cell lysates of E. coli followed by centrifugation. The enzymes were active over a broad range of temperatures, from 42°C to at least 77°C, with the highest activities observed at 77°C. R.TneDI cleaved at the center of the recognition sequence (CG↓CG) and generated blunt-end cuts. Overexpression of R.TneDI in BL21(DE3) was confirmed by both SDS-PAGE and Western blotting.

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