| 摘要: |
| 【目的】利用大肠杆菌表达纯化嗜热脱铁去硫弧菌（Deferribacter desulfuricans）解旋酶DePif1，并对其结合与解旋DNA的活性进行分析，为Pif1家族解旋酶结构和功能的阐明奠定基础。【方法】将促溶标签SUMO编码序列和人工合成的DePif1解旋酶编码序列依次连入pET15b载体，获得重组融合表达载体pET15b-SUMO-DePif1，然后将其导入E.coli BL21(DE3)菌株进行诱导表达；利用Ni-NTA亲和层析柱获得融合蛋白，SUMO蛋白酶酶切去除融合标签，再经Heparin和Ni-NTA柱分离获得无标签的纯化重组DePif1蛋白；采用荧光各向异性分析，研究pH和NaCl浓度对DePif1与DNA结合的影响及DePif1与不同底物（单链DNA、双链DNA和G4-DNA）的结合特性；使用基于荧光共振能量转移的stopped-flow技术，分析DePif1对不同底物（G4-DNA with 5′ 26 nt tail和dsDNA with 5′ 26 nt tail）的解旋活性。【结果】每升菌液可获得9 mg纯度大于95%的DePif1解旋酶。DePif1结合不同DNA底物的强度依次为G4-DNA>单链DNA>双链DNA，其对G4-DNA的解旋活力大于双链DNA。【结论】成功表达并纯化了嗜热脱铁去硫弧菌Pif1解旋酶，并证明其具有特异的G4-DNA结合和解旋能力。 |
| 关键词: 嗜热脱铁去硫弧菌 Pif1解旋酶 G4-DNA 表达纯化 解旋活性 |
| DOI： |
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| 基金项目:国家自然科学基金项目（31370798，11304252） |
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| Expression,purification and activity analysis of Pif1 helicase from thermophile Deferribacter desulfuricans |
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ZHAO Zheng-yang,LIU Na-nü,LI Hai-hong,et al
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| Abstract: |
| 【Objective】This study expressed and purified the Pif1 helicase of thermophile Deferribacter desulfuricans in E.coli and analyzed its DNA binding and unwinding activity to lay foundation for the elucidation of the structure and function of Pif1 family helicases.【Method】Based on the expression vector pET15b,a recombinant plasmid pET15b-SUMO-DePif1 was constructed by cloning the SUMO coding sequence and the synthetic DePif1 gene into the downstream of its His-tag.The recombinant plasmid was transformed into E.coli strain BL21(DE3) and the fusion DePif1 was induced by IPTG.Firstly,DePif1 with His6-SUMO tag from the supernatant of cell lysates was captured by affinity chromatography with a Ni-NTA column.Then,the fusion tag was cleaved by SUMO protease and tag-free DePif1 helicase was obtained by further purification with Heparin Sepharose Fast Flow and Ni-NTA chromatography.Finally,the effects of pH and NaCl concentrations on DNA binding,the characteristics of DePif1 binding with different substrates (single stranded DNA,double-stranded DNA,and G4-DNA) and unwinding activities of DePif1 against different substrates (G4-DNA with 5′ 26 nt tail and ds DNA with 5′ 26 nt tail) were analyzed by fluorescence anisotropy and stopped-flow based FRET.【Result】According to the above protocol,9 mg DePif1 with >95% purity was obtained for every liter of broth.DePif1 can bind with different DNA substrates with the binding activity in decreasing order of G4-DNA>ss DNA>ds-DNA.Its unwinding activity to G4-DNA was higher than to ds-DNA.【Conclusion】DePif1 helicase was expressed and purified successfully.DePif1 can bind and unwind G4-DNA specially. |
| Key words: Deferribacter desulfuricans Pif1 helicase G4-DNA expression and purification activity analysis |
