基于甲基化反应系统检测SETD3甲基化组蛋白的位点  

Detection the histone methylation by SETD3 based on the methylation reaction

作　　者：钟玙沄[1] 黄梦怡[1] 孙江[1] 黄穗[1] 李月[1] 罗海华[1] 姜勇[1] 刘靖华[1] 

机构地区：[1]广东省功能蛋白质组学重点实验室,南方医科大学基础医学院病理生理学教研室,广东广州510515

出　　处：《生物技术》2017年第3期245-251,共7页Biotechnology

基　　金：国家自然科学基金项目(“甲基化转移酶SETD4调控炎症因子释放的分子机制研究”,No.81471901);广东省自然科学基金重点项目(“甲基化转移酶SETD4在内毒素休克中的作用探讨”,No.2015A030311031)

摘　　要：[目的]构建pEGX-4T-1-Setd3,表达并纯化GST-SETD3蛋白;用获得的蛋白进行体外甲基化反应并鉴定其活性,建立体外甲基化反应系统。[方法]PCR扩增小鼠全长Setd3,插入到pEGX-4T-1载体获得pEGX-4T-1-Setd3质粒。将质粒转化大肠杆菌,用异丙基-β-D-硫代半乳糖苷(IPTG)诱导表达GST-SETD3蛋白并进行纯化。将获得的GST-SETD3蛋白定量后进行体外组蛋白甲基化反应,用Western Blot检测GST-SETD3对组蛋白H3K4及H3K36的甲基化情况。[结果]p GEX-4T-1-Setd3质粒构建正确,纯化的GST-SETD3蛋白纯度较高;甲基化反应结果表明,用不同来源的组蛋白、反应时间、反应缓冲液及检测方法均检测不到GST-SETD3二甲基化H3K4;GSTSETD3可以二甲基化H3K36。[结论]成功表达和纯化GST-SETD3蛋白;该蛋白可以使H3K36发生二甲基化,但不能使H3K4二甲基化。成功构建体外甲基化反应系统。[ Objective] To obtain SETD3 protein and identify its methyhransferase activity by in vitro assay of histone methyl- ation reaction. [ Methods] Mouse Setd3 gene was amplified by PCR and inserted into pEGX -4T - 1 vector to obtain pEGX - 4T - 1 - Setd3 plasmid. The constructed plasmid was transformed into E. coli and induced by isopropyl - 13 - D - thiogalactoside （IPTG） . Following GST - SETD3 protein purification and quantitation, the methylation of GST - SETD3 methyhransferase to H3K4 and H3K36 was verified by Western Blot . [Results] pGEX-4T- 1 -Setd3 plasmid was successfully constructed and the GST - SETD3 protein was well purified. It was found that GST - SETD3 could not methylate H3K4 whatever histones from different sources were applied, or reaction time was longer, or different buffers and methods were used, but it could methylate H3K36. [ Conclusion ] The fusion protein GST -SETD3 was successfully prepared, and it could specifically towards dimethyla- tion of H3K36, but not dimethylation of H3K4. In vitro methylation system was constructed successfully.

关 键 词：GST-SETD3 重组蛋白原核表达 蛋白纯化 甲基化反应 甲基转移酶 

分 类 号：Q518.4[生物学—生物化学] Q786