Hou Qinghua, Song Shuliang, Liang Hao, Wang Weili, Ji Aiguo
Weihai International Biotechnology R&D Center, Shandong University, Weihai 264209, China.
Se Pu. 2013 Feb;31(2):151-4. doi: 10.3724/sp.j.1123.2012.10004.
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%.
增强型绿色荧光蛋白（EGFP）是一种常用的生物标志物。在本研究中，在成功克隆和表达EGFP的基础上，建立了一种两步色谱法来分离和纯化EGFP，该方法依次包括使用HisTrap HP固定化金属亲和色谱（IMAC）和Sephadex G-10 HR尺寸排阻色谱。使用Sephacryl S-300 HR尺寸排阻色谱和十二烷基硫酸钠-聚丙烯酰胺凝胶电泳（SDS-PAGE）来检测EGFP的纯度。最后，通过荧光分光光度检测和非变性聚丙烯酰胺凝胶电泳（Native-PAGE）检测发现EGFP仍具有荧光活性。该方法可以有效地分离活性EGFP。所获得的EGFP纯度超过98%。
