gst鄄h2ax 表达载体的构建及融合蛋白的纯化 - 生命科学研究.pdfVIP

17 卷 6 期 生命科学研究 灾燥造援员7 晕燥援6 圆园13 年 12 月 蕴蚤枣藻 杂糟蚤藻灶糟藻 砸藻泽藻葬则糟澡 Dec. 圆园13 GST H2AX 表达载体的构建及融合蛋白的纯化 亮, 黄 雷, 廖晓东* (上海交通大学 医学院病理生理学教研室, 中国上海 200025) 摘 要: H2AX 属组蛋白H2A 家族成员, 其磷酸化是细胞对DNA 损伤做出反应的早期事件之一, 在启动DNA 修复过程中发挥重要功能. 利用原核表达载体pGEX鄄4T鄄1 构建了GST鄄H2AX 融合蛋白表达载体, 导入大肠杆 菌DE3 后经IPTG 诱导和GST (Glutathione S鄄transferase)磁珠纯化获得GST鄄H2AX 融合蛋白. 进一步利用GST 和H2AX 抗体对融合蛋白进行了验证. 既建立了高效 稳定的GST 蛋白纯化方法, 也为进一步研究H2AX 构 及生物学功能奠定了基础. 关键词: H2AX; 谷胱甘肽S鄄转移酶(GST); 原核表达; 融合蛋白 中图分类号: Q鄄331 文献标识码: A 文章编号: 1007鄄7847(2013)06鄄0471鄄05 Purification of H2AX Proteins Fused to Glutathione S transferase ZHANG Liang, HUANG Lei, LIAO Xiao鄄dong* (Department of Pathop hys iology, Shanghai Jiao Tong Unive rs ity School of Medic ine, Shanghai 200025, China) Abstract: H2AX is a core histone H2A variant. Rapid H2AX phosphorylation is one of the key initiating events for DNA repair in response to DNA damage. In this study, an expression system for the glutathione S鄄 transferase (GST)鄄H2AX fusion protein by using the prokaryotic expression vector pGEX鄄4T鄄1was established. The fusion protein was expressed in E. coli (DE3) through IPTG induction, and purified using GST beads. Furthermore, the identity of the fusion protein was verified by Western blot using anti鄄GST and anti鄄H2AX antibodies. In conclusion, an efficient and stable method to purify GST鄄H2AX was established, which could serve as the foundation for elucidating the structure and function of H2AX. Key words: H2AX; GST (Glutathione S鄄transferase); prokaryotic expression; fusion protein （L ife Sc ience Re