Recombinant expression, purification, and characterization of XorKII: a restriction endonuclease from Xanthomonas oryzae pv. oryzae.
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 3.5mg 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 10mM Tris-HCl pH 7.0, 10 mM MgCl(2), 1mM 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 24h.
Moon
Won Jae
WJ
Department of Chemistry, Sejong University, 98 Gunja-Dong, Gwangjin-Gu, Seoul, 143-747, [corrected] Republic of Korea.
Cho
Jae-Yong
JY
Chae
Young Kee
YK
D002021:Buffers; D003001:Cloning, Molecular; D004262:DNA Restriction Enzymes / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism*; D004587:Electrophoresis, Agar Gel; D010957:Plasmids / Q000235:genetics; D020836:Protein Structure, Quaternary; D011994:Recombinant Proteins / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism*; D014974:Xanthomonas / Q000201:enzymology*
D002021:Buffers; D011994:Recombinant Proteins; D004262:DNA Restriction Enzymes