核心组蛋白的融合表达和纯化  

Fusion Expession and Purification of Nucleosmal Histones

作　　者：曹佳[1,2] 丁丽华[1] 范忠义[1] 程龙[1] 蒋凯[1] 徐晓洁[1] 韩永健[1] 徐承水[2] 叶棋浓[1] 

机构地区：[1]军事医学科学院生物工程研究所,北京100850 [2]曲阜师范大学生命科学学院,山东曲阜273165

出　　处：《生物技术通讯》2011年第4期484-487,共4页Letters in Biotechnology

基　　金：国家自然科学基金(30870499;31071174;81072173)

摘　　要：目的:克隆核心组蛋白H2A、H2B、H3和H4的基因,表达并纯化组蛋白与谷胱甘肽S-转移酶(GST)的融合蛋白。方法:用PCR方法从乳腺文库中扩增核心组蛋白H2A、H2B、H3和H4的编码序列,分别将其以正确相位与pGEX-KG载体中的GST编码序列融合,得到重组质粒pGST-H2A、pGST-H2B、pGST-H3和pGST-H4,分别转化大肠杆菌BL21,表达融合蛋白GST-H2A、GST-H2B、GST-H3和GST-H4;用谷胱甘肽-Sepharose 4B亲和纯化融合蛋白;用Western印迹检测融合蛋白的表达及纯化。结果:分别构建了核心组蛋白H2A、H2B、H3和H4的融合表达载体;Western印迹检测表明,融合蛋白GST-H2A、GST-H2B、GST-H3和GST-H4获得表达及纯化。结论:表达并纯化了H2A、H2B、H3和H4的融合蛋白,为进一步研究核心组蛋白的功能奠定了基础。Objective： To clone,express and purify the nucleosmal histone family members H2A,H2B,H3 and H4.Methods： The coding sequences of H2A,H2B,H3 and H4 were amplified by PCR and fused in frame with the coding region of GST in the pGEX-KG vector to generate the pGST-H2A,pGST-H2B,pGST-H3 and pGST-H4 recombinant plasmids.The GST-H2A,GST-H2B,GST-H3 and GST-H4 fusion proteins were expressed in E.coli BL21 and purified by glutathione-Sepharose 4B affinity chromatography.The fusion proteins were characterized by Western blot.Results： The recombinant plasmids were successfully constructed.Western blot analysis showed that the fusion proteins were expressed.Conclusion： The genes coding H2A,H2B,H3 and H4 were successfully cloned and their fusion proteins were purified.This lays a solid foundation for further study on the function of nucleosmal histones.

关 键 词：组蛋白 克隆 表达 融合蛋白 

分 类 号：Q78[生物学—分子生物学]