Abstract:Abstract: 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 kdm5b 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.
引用本文:
王少杰, 王春喜, 韩 伟. 乳腺癌靶标蛋白KDM5B的表达及纯化[J]. 生命科学研究, 2016, 20(6): 502-509.
WANG Shao-Jie, WANG Chun-Xi, HAN Wei. Expression and Purification of Breast Cancer Target Protein KDM5B. Life Science Research, 2016, 20(6): 502-509.