TfoI produced by Tepidimonas fonticaldi PL17, a moderate thermophilic bacterium, is an isoschizomer of MseI
A moderately thermophilic Gram-negative bacterium isolated from the Polok hot spring, Sikkim, India, was identified as a strain (PL17) of Tepidimonas fonticaldi by 16S rDNA sequencing. T. fonticaldi PL17 produces a Type IIP restriction endonuclease; named TfoI. Restriction mapping, run-off sequencing of TfoI-digests of dsDNA fragments, and end compatibility of TfoI with NdeI confirmed that the enzyme recognizes and cleaves the sequence 5′–T^TAA–3′, and is thus an isoschizomer of MseI. The TfoI restriction–modification genes in the T. fonticaldi PL17 genome were identified, and the annotated TfoI protein encodes a protein of 181 amino acid residues that shares 47.2% sequence identity with MseI. The native enzyme was purified using a four-column chromatography protocol, and its functional homogeneity was confirmed by standard quality control tests. The ESI-MS measured molecular weight of purified TfoI (20.696 kDa) is in agreement with that of the calculated monomeric molecular weight of the predicted TfoI protein sequence (20.694 kDa). TfoI exhibits optimal activity in the temperature range of 55–70 °C with Mg+2 or Co+2 as cofactor. Similar to its isoschizomers, TfoI can be used as the frequent cutter for genome analysis.
KeywordsTepidimonas fonticaldi Thermophile Type II restriction endonuclease Isoschizomer Chromatography
This work was supported by the financial assistance from CSIR-IMTECH (BSC0402, BSC0120 to AKP; OLP0091 to BK) and Department of Biotechnology-India (BT/PR7368/INF/22/177/2012 to AKP and GAP0096 to BK) funding to BK. The authors are thankful to Dr. Rakshak Kumar for sample collection, and acknowledge the support from the Sikkim State Council of Science and Technology and the Department of Forest, Govt. of Sikkim, in sample collection. RK is supported by the research fellowship scheme of University Grants Commission, India. BK is a recipient of the Department of Biotechnology-Ramalingaswami re-entry fellowship.
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