| 摘要: |
| 【目的】通过原核表达系统获得嗜热的厌氧棒菌（Anaerobaculum hydrogeniformans）DNA解旋酶Pif1（AnaPif1），研究其体外最佳解旋条件，为深入研究嗜热菌Pif1家族解旋酶工作机理提供参考。【方法】构建重组载体pET15b-sumo-AnaPif1，将重组载体转入表达菌株BL21(DE3)中诱导表达目的蛋白，经Ni-NTA柱和Heparin柱纯化最终获得全长AnaPif1蛋白；利用停流-荧光共振能量转移（FRET）技术，以pH值和Tris-HCl、NaCl、MgCl2浓度及反应温度、能量供体为考察因素，以反应时间常数或速率常数为指标，摸索该蛋白在体外的最佳解旋条件、最佳能量供体以及底物的偏好性。【结果】成功构建了pET15b-sumo-AnaPif1重组表达载体，通过诱导表达，获得了纯度大于95%的全长AnaPif1蛋白，其大小为55 ku，等电点为6.89。AnaPif1在体外的最佳解旋条件为：Tris-HCl (pH 7.0)20 mmol/L、NaCl 20 mmol/L、MgCl2 2 mmol/L、反应温度37 ℃；其能够利用4种核苷三磷酸（ATP/GTP/CTP/UTP）作为能量供体，但对ATP有偏好性；该酶还能解旋多种底物（双链DNA、G4-DNA），且对底物选择无明显偏好性。【结论】获得了纯化的AnaPif1解旋酶，明晰了其最佳解旋条件、能量供体及底物的偏好性特征。 |
| 关键词: 厌氧棒菌 Pif1解旋酶 蛋白表达纯化 DNA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 a Pif1 helicase from Anaerobaculum hydrogeniformans |
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GUO Hailei,LIU Nanü,DUAN Xiaolei,et al
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| Abstract: |
| 【Objective】The Pif1 helicase from Anaerobaculum hydrogeniformans (AnaPif1) was obtained by prokaryotic expression system,and the optimal conditions were explored for AnaPif1 to display unwinding activity in vitro.This research would be helpful for further study on the unwinding mechanism of Pif1 family helicases.【Method】The entire gene of AnaPif1 was synthesized and a recombinant plasmid pET15b-sumo-AnaPif1 was constructed.Then,it was transformed into Escherichia coli host strain BL21(DE3).The full-length AnaPif1 helicase was purified through Ni-NTA affinity chromatography followed by Heparin chromatography with fast protein liquid chromatography(FPLC) system.The stopped-flow and fluorescence resonance energy transfer (FRET) technique were used to investigate the effect of pH value and concentrations of Tris-HCl,NaCl,MgCl2 as well as different temperature,energy donors and substrates on reaction activity of AnaPif1 in vitro.The time constant or rate constant were used as the determinant for optimal unwinding conditions for AnaPif1,selectivity of DNA substrates and energy donors. 【Result】The recombinant plasmid pET15b-sumo-AnaPif1 was successfully constructed and the full-length AnaPif1 helicase with high purity (>95%) was obtained.The optimal unwinding conditions in vitro were Tris-HCl (pH 7.0) 20 mmol/L,NaCl 20 mmol/L,MgCl2 2 mmol/L at 37 ℃.All nucleoside triphosphates could act as its energy donors,while ATP was preferred.AnaPif1 had no preference for DNA substrates such as G4-DNA and double-stranded DNA.【Conclusion】AnaPif1 helicase was obtained,and the optimal unwinding conditions in vitro,energy donors and the preference of DNA substrates were determined. |
| Key words: Anaerobaculum hydrogeniformans Pif1 helicase expression and purification of protein DNA unwinding activity |
