热脱硫弧菌解旋酶基因TyPif1的原核表达、纯化及功能分析

Prokaryotic expression,purification and functional analysis of Thermodesulfovibrio yellowstonii H.TyPif1

【目的】通过原核表达系统获得热脱硫弧菌（Thermodesulfovibrio yellowstonii H.）Pif1解旋酶（TyPif1）,研究TyPif1解旋酶的嗜热特性和解旋机理。【方法】将Typif1解旋酶编码区和SUMO促溶标签编码区融合连入pET15b获得重组表达载体pET15b-SUMO-TyPif1,将该重组载体导入大肠杆菌BL21（DE3）菌株诱导表达,经Ni-NTA柱亲和纯化获得融合蛋白,SUMO蛋白酶切除标签,再经Ni-NTA柱去除标签蛋白及SUMO蛋白酶,经Heparin柱进一步纯化最终获得TyPif1全长蛋白。通过荧光各向异性、圆二色谱（CD）和荧光共振能量转移（FRET）分析TyPif1解旋酶的DNA结合活性、解旋活性以及二级结构的热稳定性。【结果】获得了纯度大于95%的TyPif1蛋白,1L培养基可以获得约10mg纯度大于95%的蛋白;TyPif1解旋酶结合单链DNA和G4DNA的活性明显高于双链DNA;TyPif1具有较高的解旋G4DNA的活性,在25~60℃下其二级结构相对稳定,且解旋速率随温度升高而增大,25~50℃TyPif1的解旋速率由0.09s-1增大到0.23s-1。【结论】表达纯化了热脱硫弧菌TyPif1解旋酶,其不仅具有良好的热稳定性,同时具有特异的结合和解旋G4DNA的能力。

【Objective】This research used prokaryotic expression system to obtain Pif1 helicase from Thermodesulfovibrio yellowstonii H.（TyPif1）,and studied the mechanism of thermophilic characteristic and unwinding mechanism of TyPif1 helicase.【Method】The Pif1 gene of Thermodesulfovibrio yellowstonii H.（Typif1）and a SUMO protein folding tag gene were synthesized to pET15 bto construct recombinant plasmid pET15b-SUMO-TyPif1.Then,the plasmid was transformed into Escherichia coli host strain BL21（DE3）,and the TyPif1 helicase was purified by two-step Ni-NTA affinity chromatography and onestep Heparin chromatography at 4 ℃ using FPLC.Fluorescence anisotropy,fluorescence resonance energy transfer（FRET）and circular dichroism spectrum（CD）were used to analyze the DNA binding and unwinding activity of TyPif1 as well as the stability of its secondary structure.【Result】The full-length TyPif1 helicase with high purity of95% was obtained.Its binding activity with ssDNA and G-quadruplex（G4）DNA was much higher than that of dsDNA.TyPif1 has high activity to unwound G4 DNA.The secondary structure was relatively stable under 25-60℃,and the unwinding rate increased from 0.09s-1 to 0.23s-1with the increase of temperature from 25 to 50 ℃.【Conclusion】TyPif1helicase was expressed and purified.TyPif1 helicase not only had good thermal stability,but also had specific ability of binding and unwinding G4 DNA.