XspI, a new type II restriction endonuclease from a Xanthomonas species.
A new type II restriction endonuclease, XspI, was purified 11-fold in seven steps to homogeneity from Xanthomonas sp. strain YK1 grown aerobically in Luria broth. The final specific activity of the purified enzyme was 3890 micrograms of lambda DNA digested per h per mg of protein. The molecular weight of the native enzyme was determined to be 54,000. Sodium dodecyl sulfate-gel electrophoresis revealed two identical subunits of molecular weight 29,000. The purified enzyme was most active at 37 degrees C in the presence of 100 mM KCl and 5 mM MgCl2 under pH range from 7.5 to 8.0. The enzyme was stable at least for 1 h at 37 degrees C, but was inactivated after 20 min at 65 degrees C. XspI recognized the tetranucleotide sequence, 5'-CTAG-3', and cleaved it between C and T, like its isoschizomers MaeI and BfaI. The ability of the source organism to grown aerobically, together with the heat inactivation of the enzyme, confers practical advantages upon XspI over its known isoschizomers.
Song
T
T
Department of Biology, Yonsei University, Seoul, Korea.
Sik Kang
B
B
Min Kim
Y
Y
D001483:Base Sequence; D001665:Binding Sites; D015252:Deoxyribonucleases, Type II Site-Specific / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism; D008970:Molecular Weight; D009841:Oligonucleotides / Q000737:chemistry / Q000378:metabolism; D011487:Protein Conformation; D012988:Soil Microbiology; D013379:Substrate Specificity; D014974:Xanthomonas / Q000201:enzymology*
D009841:Oligonucleotides; C040836:CTAG-specific type II deoxyribonucleases; D015252:Deoxyribonucleases, Type II Site-Specific