组蛋白去甲基化酶JARID1B的表达纯化及酶活力分析

摘要

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.%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-rich interactive 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 of flight 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10 mg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.