Chinese Journal of Chromatography

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

• Technical Notes • Previous Articles     Next Articles

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

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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)