基础医学与临床 ›› 2010, Vol. 30 ›› Issue (7): 721-725.

• 研究论文 • 上一篇    下一篇

人PHF8蛋白的原核表达、纯化与结晶

黄硕 余琳 汪洋 刘音 陈忠周   

  1. 中国医学科学院基础医学研究所医学分子生物学国家重点实验室 中国医学科学院基础医学研究所 中国农业大学生物学院农业生物技术国家重点实验室
  • 收稿日期:2010-04-07 修回日期:2010-05-04 出版日期:2010-07-05 发布日期:2010-07-05
  • 通讯作者: 陈忠周

Prokaryotic expression, purification and crystallization of human PHF8

Shuo HUANG, Lin YU, Yang WANG, Yin LIU, Zhong-zhou CHEN   

  1. Institute of Basic Medical Sciences, CAMS and PUMC Institute of Basic Medical Sciences, CAMS and PUMC College of Biological Sciences, China Agricultural University
  • Received:2010-04-07 Revised:2010-05-04 Online:2010-07-05 Published:2010-07-05
  • Contact: Zhong-zhou CHEN

摘要: 目的 探讨人PHF8蛋白的原核表达,纯化与结晶条件。方法 用分子克隆的方法构建重组质粒PHF8/pet28b,测序鉴定正确后将其转入Rosetta 2(DE3),IPTG诱导表达融合蛋白。对融合蛋白进行分离纯化结晶,衍射分析,并用计算机软件解析其晶体结构。结果 经过诱导,含有PHF8/pet28b的Rosetta 2在原核表达系统中表达出了相对分子质量为40kd的c-PHF8融合蛋白,c-PHF8在合适的悬滴条件下得到六方柱晶体。结论 利用原核表达体系成功地表达了人PHF8催化活性中心结构域并获得结晶,为进一步的三维结构解析和PHF8功能研究奠定了基础。

Abstract: Objective Purification and crystallization of human PHF8. Methods The plasmid PHF8/pet28b, constructed by molecular cloning, was transformed into Rosetta2 (DE3) cell, and PHF8 was induced by IPTG. Recombinant protein was purified, crystallized, and diffracted and its structure was solved. Result Recombinant PHF8 protein was expressed in E. coli, and subjected to affinity purification followed by gel filtration separation. PHF8 protein with hexagon-1ike crystals was obtained. Conclusion The enzymatically active PHF8 protein was purified using the prokaryotic expression system, its crystal particle was obtained, and the overall structure of PHF8 catalytic core was resolved.