| 摘要: |
| 【目的】探索牛和果蝇DHX36解旋酶的表达纯化条件及底物结合活性，为深入研究DEAH-box解旋酶提供理论参考。【方法】以较高等动物牛(Bos taurus)和较低等动物黑腹果蝇(Drosophila melanogaster)RNA解旋酶BtDHX36和DmDHX36为研究对象,首先，构建重组表达载体pET15b-sumo-BtDHX36和pET15b-sumo-DmDHX36，将2种质粒分别转入表达菌株BL21(DE3)中,诱导表达2种目的蛋白，利用Ni-NTA层析柱、HisTrap SP HP柱纯化得到目的蛋白。然后，采用荧光各向异性法(FA)研究溶液和温度条件对2种蛋白底物结合活性的影响，以及二者的底物结合偏好性。最后利用快速停流-荧光共振能量转移(FRET)技术对2种DHX36的双链底物解旋活性进行比较。【结果】获得了纯度大于95%的全长BtDHX36和DmDHX36蛋白，2种DHX36解旋酶的最佳底物结合条件为：20 mmol/L Tris-HCl （pH 7.5）、50 mmol/L NaCl、2 mmol/L MgCl2、反应温度37 ℃。在此条件下，2种同源DHX36均可结合多种类型核酸底物(ssRNA、ssDNA、parallel G4、anti parallel G4)，且BtDHX36结合平行结构G4底物(parallel G4)的倾向更明显，DmDHX36无此趋势；2种DHX36解旋酶结合不同长度ssDNA的能力不同，BtDHX36更倾向于结合较长的单链DNA底物，而DmDHX36更倾向于结合较短的单链DNA底物；二者均能高效解旋dsRNA和dsDNA底物，DmDHX36更倾向于解旋dsDNA，而BtDHX36无明显倾向性。【结论】成功表达并纯化了BtDHX36和DmDHX36蛋白，确定了其最适结合条件，比较了2种DHX36解旋酶对不同底物的结合及解旋偏好。 |
| 关键词: 牛DHX36解旋酶 果蝇DHX36解旋酶 蛋白表达纯化 底物结合 底物解旋 |
| 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 comparison of DHX36 helicase between Bos taurus and Drosophila melanogaster |
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LI Kang,GUO Hailei,XI Xuguang
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| Abstract: |
| 【Objective】This study explored the conditions for expression,purification and substrate binding activity of DHX36 helicase of cattle and drosophila to provide reference for further study of DEAH-box helicase.【Method】RNA helicases BtDHX36 and DmDHX36 of higher animal cattle (Bos taurus) and lower animal drosophila (Drosophila melanogaster) were selected for recombinant expression vectors pET15b-sumo-BtDHX36 and pET15b-sumo-DmDHX36.The two plasmids were transferred into the expression strain BL21(DE3) to induce expression of proteins.Target proteins were purified by a Ni-NTA column and a HisTrap SP HP column.Then,the effect of solution and temperature conditions on the binding ability of the two protein substrates and the substrate binding preferences were studied by fluorescence anisotropy (FA).Finally,the double chain desorption ability of DHX36 was compared and analyzed using fast stop current fluorescence resonance energy transfer (FRET) technique.【Result】Full length BtDHX36 and DmDHX36 proteins with greater than 95% purity were obtained.The influence of solution and temperature conditions on the binding activity of the two DHX36 substrates was determined,and the optimal substrate binding conditions were 20 mmol/L Tris-HCl(pH 7.5),50 mmol/L NaCl,2 mmol/L MgCl2,and reaction temperature 37 ℃.Under these conditions, both homologous DHX36 were bind to various nucleic acid substrates (ssRNA,ssDNA,parallel G4 and anti parallel G4),and the tendency of BtDHX36 to bind parallel G4 substrate (parallel G4) was more obvious while DmDHX36 had no such trend.The ability to bind ssDNA with different lengths was different.BtDHX36 was more likely to bind longer single stranded DNA substrates,while DmDHX36 was more likely to bind shorter single-stranded DNA substrates.Both can efficiently unwind dsRNA and dsDNA substrates.DmDHX36 was more inclined to unwind dsDNA,while BtDHX36 had no tendency.【Conclusion】The BtDHX36 and DmDHX36 proteins were successfully expressed and purified,and the optimal binding conditions were determined.The binding and unwinding preferences of two DHX36 substrates to different substrates were also compared. |
| Key words: Bos taurus DHX36 helicase Drosophila melanogaste DHX36 helicase protein expression purification substrate binding substrate unwinding |
