Cloning and characterization of filamentous temperature-sensitive protein Z from Xanthomonas oryzae pv. Oryzae.
Cloning and characterization of filamentous temperature-sensitive protein Z from Xanthomonas oryzae pv. Oryzae.
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米黄单胞菌丝状温度敏感蛋白 Z 的克隆和表征。
DOI：
10.1186/s40064-016-1876-3
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发表时间：
2016
期刊：
影响因子：
--
通讯作者：
Long ZE
中科院分区：
文献类型：
--
作者：
Dai L;Huang Y;Chen Y;Long ZE
黄单胞菌黄萎病菌ftsZ基因利用特异性引物进行PCR扩增，成功构建了重组质粒pET-22 b-ftsZ。将带有6× His标签的FtsZ在大肠杆菌BL 21中以可溶性形式过量表达，并通过Ni-NTA琼脂糖柱纯化。纯化的重组FtsZ在SDS-PAGE上显示单一条带，表观分子量约为44 kDa，并通过western blotting分析证实。重组FtsZ的最适温度为50 °C，最适pH为7.0。重组FtsZ显示出良好的稳定性，并且在50 °C下保持> 95%的活性240分钟。GT3酶活性符合米氏动力学，Km = 1.750 mM，Vmax = 0.155 nmol Pi/min/nmol FtsZ。
The ftsZ gene from Xanthomonas oryzae pv. Oryzae was amplified by PCR with the specific primers, and the recombinant plasmid pET-22b-ftsZ was constructed successfully. The FtsZ with a 6× His tag was overexpressed in a soluble form in Escherichia coli BL21 and purified through a Ni-NTA agarose column. The purified recombinant FtsZ showed a single band on SDS-PAGE with an apparent molecular mass of about 44 kDa, and confirmed by western blotting analysis. The optimum temperature for GTPase activity of the recombined FtsZ was 50 °C, and the optimum pH was 7.0. The recombinant FtsZ showed good stability and retained >95 % activity at 50 °C for 240 min. The GTPase activity followed Michaelis–Menten kinetics with the KM of 1.750 mM and the Vmax of 0.155 nmol Pi/min/nmol FtsZ respectively.