【摘要】 目的筛选能够大量表达、方便纯化且具有酶活性的组蛋白去甲基化酶JARID1B(jumonji AT-richinteractive domain 1B)的截短体。方法通过Bac-to-Bac系统制备含目的基因的杆粒(bacmid),转染Sf9昆虫细胞获得重组病毒基因组,并感染Hi5昆虫细胞表达目的蛋白,用Ni-NTA亲和层析,离子交换层析和分子筛纯化目的蛋白。利用基质辅助激光解析电离飞行时间质谱技术(matrix-assisted laser desorption ionization time offlight mass spectrometer,MALDI-TOF)分析截短体的酶活力。结果去除C-端两个PHD结构域的截短体JARID1B1-825能够在昆虫细胞Hi5中得以大量表达(1L细胞含10mg蛋白),经Ni-NTA亲和层析,阴离子交换层析和分子筛三步纯化,获得纯度大于90%的目的蛋白,MALDI-TOF质谱分析显示JARID1B1-825仍拥有去组蛋白甲基化修饰的酶活性。结论筛选到了能够大量表达、方便纯化且具有酶活性的JARID1B的截短体,对其后续的功能研究和药物开发具有重要的意义。更多还原

【Abstract】 Objective To obtain high-quality truncated human histone demethylase JARID1B(jumonji AT-rich interactive domain 1B) in large amount.Methods We used Bac-to-Bac baculovirus expression system to express proteins followed by Ni-NTA affinity chromatography,ion exchange and gel filtration.Matrix-assisted laser desorption ionization time of flight mass spectrometer(MALDI-TOF) was employed to analyse the demethylation activity.Results JARID1B1-825,a construct lack of the C-terminal domain,was able to be expressed in Hi5 insect cells in great quantity.The yield was 10 mg protein per liter insect cell culture.In vitro demethylase activity analysis of JARID1B1-825 showed that this truncated JARID1B1-825 possessed the demethylation activity.Conclusions Large amount of JARID1B with hight-quality was obtained.This research will be useful for future structural and functional studies of JARID1B and related drug screening.更多还原