Journal
PROTEIN EXPRESSION AND PURIFICATION
Volume 62, Issue 2, Pages 230-234Publisher
ACADEMIC PRESS INC ELSEVIER SCIENCE
DOI: 10.1016/j.pep.2008.08.007
Keywords
XorKII; Restriction endonuclease; Xanthomonas; Recombinant
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Funding
- RDA Biogreen21 Fund [20050401-034-743-176-06-00]
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An endonuclease from Xanthomonas oryzae pathovar oryzae (Xoo) KACC10331, XorKII, was recombinantly produced in Escherichia coli by applying the stationary state induction method, which was necessary to prevent the unwanted lysis of E. coli cells. XorKII was purified by immobilized metal affinity chromatography on an FPLC system. The yield was 3.5 mg of XorKII per liter of LB medium. The purified recombinant XorKII showed that it recognized and cleaved to the same site as PstI. It behaved as a dimer as evidenced by the size exclusion chromatography. The specific activity of the purified XorKII was determined to be 31,300 U/mg. The enzyme activity was monitored by cleaving lambda DNA or YEp24 plasmid as substrates. The enzyme was the most active at 10 mM Tris-HCl pH 7.0, 10 mM MgCl2, 1 mM dithiothreitol at 37 degrees C. XorKII was easily inactivated by heating at 65 degrees C for 5 min, but retained most of the original activity after incubation at 37 degrees C for 24 h. (c) 2008 Elsevier Inc. All rights reserved. In this work, we focused on XorKII, one of the two restriction
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