A rapid purification procedure for the HsdM protein of EcoR124I and biophysical characterization of the purified protein |
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| Institution: | 1. Department of Biology, Faculty of Science, University of Guilan, Rasht, Iran;2. Bioinformatics and Computational Omics Lab (BioCOOL), Department of Biophysics. Faculty of Biological Sciences, Tarbiat Modares University, Tehran, Iran;3. Department of Quantitative Health Sciences, John A. Burns School of Medicine, University of Hawaii at Manoa, Honolulu, HI 96813, USA;4. Department of Molecular Biotechnology, Cell Science Research Center, Royan Institute for Biotechnology, ACECR, Isfahan, Iran;5. Department of Molecular and Cellular Sciences, Faculty of Advanced Sciences and Technology, pharmaceutical Sciences Branch, Islamic Azad University, Tehran, Iran (IAUPS);6. Department of Biochemistry, Erasmus University Medical Center, P.O. Box 2040, 3000 CA Rotterdam, The Netherlands;7. Department of Biology, Payame Noor University, Tehran, Iran;8. Pediatric Cell Therapy Research Center, Children''s Medical Center, Tehran University of Medical Sciences, Tehran, Iran |
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| Abstract: | Type I restriction–modification (R–M) systems are comprised of two multi-subunit enzymes with complementary functions: the methyltransferase (∼160 kDa), responsible for methylation of DNA, and the restriction endonuclease (∼400 kDa), responsible for DNA cleavage. Both enzymes share a number of subunits, including HsdM. Characterisation of either enzyme first requires the expression and purification of its constituent subunits, before reconstitution of the multisubunit complex. Previously, purification of the HsdM protein had proved problematic, due to the length of time required for the purification and its susceptibility to degradation. A new protocol was therefore developed to decrease the length of time required to purify the HsdM protein and thus prevent degradation. Finally, we show that the HsdM subunit exhibits a concentration dependent monomer–dimer equilibrium. |
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