摘要
组蛋白修饰是表观遗传重要的调控机制之一,在基因表达调控、异染色质形成等生命过程中起着重要作用。组蛋白去甲基化酶lysine demethylation 5B(KDM5B)是参与组蛋白修饰的一种重要蛋白质,Jumonji C(Jmj C)是其催化结构域,且与乳腺癌密切相关。现以kdm5b基因为模板,通过PCR分别扩增Jumonji N(Jmj N)、Jmj C结构域片段,用5×GS linker连接,插入到原核表达载体pGEX-6p-1,转化至Rosetta(DE3)感受态细胞并表达,重组蛋白经亲和柱GSTrap、分子筛superdexG75和阴离子交换柱Resource Q纯化后,再进行圆二色谱和体外酶活实验。SDS-PAGE、圆二色谱和MALDI-TOF质谱分析结果表明,成功获得纯度较高的重组蛋白,且具有去甲基化的活性。以上结果为进一步基于Jmj C结构域进行小分子抑制剂筛选奠定了基础。
Histone modification as one of the key epigenetic regulation mechanisms plays a critical role in various biological processes,such as gene expression regulation and heterochromatin formation.Histone demethylase lysine demethylation 5B（KDM5B） is a significant protein involved in histone modification by catalytic domain Jumonji C（JmjC） and closely related with breast cancer.Here,DNA fragments of Jumonji N（JmjN） and JmjC domains were amplified by PCR respectively using kdm5 b gene as template,connected with 5× GS linker,and cloned into the prokaryotic expression vector pGEX-6p-1.The constructed plasmid was transformed into competent cell Rosetta（DE3） and expressed.Then,the recombinant fusion protein was purified by affinity chromatography GSTrap,molecular exclusion chromatography superdex G75 and anion exchange chromatography Resource Q,followed by circular dichroism（CD） and demethylation activity analysis in vitro.Results of SDS-PAGE,CD and MALDI-TOF showed that the recombinant protein was obtained successfully with high purity and possessed demethylation activity.These results will be the foundation for further study of small molecule inhibitor screening based on JmjC domain.
出处
《生命科学研究》
CAS
CSCD
2016年第6期502-509,共8页
Life Science Research
基金
上海市科学技术委员会科研计划项目(15ZR1449300)
