一种嗜热菌Pif1解旋酶的表达纯化及活性分析  

Expression,purification and activity analysis of Pif1 helicase from thermophile Deferribacter desulfuricans

作　　者：赵正阳[1] 刘娜女[1] 李海红[1] 奚绪光[1] 范三红[1] 

机构地区：[1]西北农林科技大学生命科学学院,陕西杨凌712100

出　　处：《西北农林科技大学学报（自然科学版）》2016年第1期169-176,共8页Journal of Northwest A&F University(Natural Science Edition)

基　　金：国家自然科学基金项目(31370798;11304252)

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′26nt tail和dsDNA with 5′26nt 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。[Objective] This study expressed and purified the Pifl helicase of thermophile Deferribacter desulfuricans in E. coli and analyzed its DNA binding and unwinding activity to lay foundation for the elu- cidation of the structure and function of Pifl family helicases. [Method] Based on the expression vector pET15b,a recombinant plasmid pET15b-SUMO-DePif1 was constructed by cloning the SUMO coding se- quence and the synthetic DePifl gene into the downstream of its His-tag. The recombinant plasmid was transformed into E. coli strain BL21 （DE3） and the fusion DePifl was induced by IPTG. Firstly, DePifl 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 DePifl helicase was ob- tained by further purification with Heparin Sepharose Fast Flow and Ni-NTA chromatography. Finally, theeffects of pH and NaC1 concentrations on DNA binding,the characteristics of DePifl binding with different substrates （single-stranded DNA,double-stranded DNA, and G4-DNA） and unwinding activities of DePifl 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 De- Pill with ~95G purity was obtained for every liter of broth. DePifl can bind with different DNA sub- strates 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] DePifl helicase was expressed and purified success- fully. DePifl can bind and unwind G4 DNA specially.

关 键 词：嗜热脱铁去硫弧菌 Pif1解旋酶 G4DNA 表达纯化 解旋活性 

分 类 号：Q71[生物学—分子生物学]