色谱 ›› 2013, Vol. 31 ›› Issue (2): 151-154.DOI: 10.3724/SP.J.1123.2012.10004

• 技术与应用 • 上一篇    下一篇

增强型绿色荧光蛋白的色谱分离和纯化

侯清华1,2, 宋淑亮1,2, 梁浩1,2, 王伟莉1,2, 吉爱国1,3*   

  1. 1. 山东大学(威海)国际生物技术研发中心, 山东 威海 264209; 2. 山东大学(威海)海洋学院, 山东 威海 264209; 3. 山东大学药学院, 山东 济南 250012
  • 收稿日期:2012-10-10 修回日期:2012-11-15 出版日期:2013-02-28 发布日期:2013-02-05
  • 通讯作者: 吉爱国

Isolation and purification of enhanced green fluorescent protein using chromatography

HOU Qinghua1,2, SONG Shuliang1,2, LIANG Hao1,2, WANG Weili1,2, JI Aiguo1,3*   

  1. 1. Weihai International Biotechnology R& D Center, Shandong University, Weihai 264209, China; 2. Marine School of Shandong University at Weihai, Weihai 264209, China; 3. Pharmaceutical School of Shandong University, Jinan 250012, China
  • Received:2012-10-10 Revised:2012-11-15 Online:2013-02-28 Published:2013-02-05

摘要: 增强型绿色荧光蛋白(EGFP)是生物领域常用的标记物。在前期成功克隆表达EGFP的基础上,本实验建立了两步分离纯化EGFP的色谱方法,并验证其分离纯化效果,检验EGFP的活性。首先用金属螯合亲和色谱柱HisTrap HP对EGFP的重组菌体破碎上清液进行初步分离,再用葡聚糖凝胶排阻色谱柱Sephadex G-10 HR对其进行脱盐纯化。采用丙烯葡聚糖凝胶排阻色谱柱Sephacryl S-300 HR和十二烷基磺酸钠-聚丙烯酰胺凝胶电泳(SDS-PAGE)检测分离纯化后的EGFP纯度。最后通过荧光分光检测器和非变性聚丙烯酰胺凝胶电泳(Native-PAGE)验证分离纯化后的EGFP是否具有荧光活性。结果表明该方法可以简便快速地分离纯化EGFP,纯度超过98%,同时保持了EGFP的荧光活性。

关键词: 纯化, 分离, 金属螯合亲和色谱, 凝胶排阻色谱, 增强型绿色荧光蛋白

Abstract: Enhanced green fluorescent protein (EGFP) is a common biological marker. In this research, on the foundation of successful clone and expression of EGFP, a two-step chromatographic method was established to separate and purify EGFP, which includes the use of HisTrap HP immobilized metal affinity chromatography (IMAC) and Sephadex G-10 HR size exclusion chromatography in sequence. Sephacryl S-300 HR size exclusion chromatography and sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) were used to check out the purity of EGFP. At last, it was found that EGFP still had fluorescent activity using fluorescence spectrophotometric detection and Native-PAGE detection. This method can effectively separate the active EGFP. The purity of the obtained EGFP was more than 98%.

Key words: enhanced green fluorescent protein (EGFP), gel exclusion chromatography (GEC), purification, separation, immobilized metal affinity chromatography (IMAC)