Molecular Biology Reports

, Volume 39, Issue 5, pp 5607–5614

Characterization of MspNI (G/GWCC) and MspNII (R/GATCY), novel thermostable Type II restriction endonucleases from Meiothermus sp., isoschizomers of AvaII and BstYI

Article

DOI: 10.1007/s11033-011-1365-2

Cite this article as:
Gupta, R., Xu, SY., Sharma, P. et al. Mol Biol Rep (2012) 39: 5607. doi:10.1007/s11033-011-1365-2

Abstract

MspNI and MspNII, isoschizomers of prototype Type II restriction endonucleases AvaII and BstYI, were extracted from an extreme thermophile bacterium belonging to the genus Meiothermus, isolated from the hot sulphur springs in north Himalayan region of India where temperature and pH ranged from 60 to 80°C and 7.5 to 8.5, respectively. The two enzymes were purified to homogeneity using Cibacron-Blue 3GA Agarose, Q-Sepharose and SP-Sepharose chromatography and were homodimers with subunit molecular weights of 27 and 45 kDa, respectively. Restriction mapping and run-off sequencing of MspNI and MspNII cleaved pBR322 DNA showed that they recognized and cleaved 5′-G/GWCC-3′ and 5′-R/GATCY-3′ sites, respectively. MspNI and MspNII worked optimally at 60 and 70°C, 6 and 5 mM MgCl2, respectively and showed no star activity in organic solvents. Both were resistant to sequence methylation and were stable up to 25 PCR cycles.

Keywords

MspNI MspNII Meiothermus Restriction endonucleases Thermostable Isoschizomer 

Supplementary material

11033_2011_1365_MOESM1_ESM.doc (738 kb)
Supplementary material 1 (DOC 738 kb)

Copyright information

© Springer Science+Business Media B.V. 2011

Authors and Affiliations

  1. 1.Department of BiotechnologyPanjab UniversityChandigarhIndia
  2. 2.Department of MicrobiologyPanjab UniversityChandigarhIndia
  3. 3.New England BiolabsIpswichUSA

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