Applied Microbiology and Biotechnology

, Volume 77, Issue 5, pp 1175–1180

Purification of green fluorescent protein using a two-intein system

  • Zhonglin Zhao
  • Wei Lu
  • Baoqing Dun
  • Dan Jin
  • Shuzhen Ping
  • Wei Zhang
  • Ming Chen
  • Ming-Qun Xu
  • Min Lin
Methods

Abstract

A two-intein purification system was developed for the affinity purification of GFPmut3*, a mutant of green fluorescent protein. The GFPmut3* was sandwiched between two self-cleaving inteins. This approach avoided the loss of the target protein which may result from in vivo cleavage of a single intein tag. The presence of N- and C-terminal chitin-binding domains allowed the affinity purification by a single-affinity chitin column. After the fusion protein was expressed and immobilized on the affinity column, self-cleavage of the inteins was sequentially induced to release the GFPmut3*. The yield was 2.41 mg from 1 l of bacterial culture. Assays revealed that the purity was up to 98% of the total protein. The fluorescence and circular dichroism spectrum of GFPmut3* demonstrated that the purified protein retains the correctly folded structure and function.

Keywords

Intein Protein splicing Green fluorescent protein Protein purification 

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Copyright information

© Springer-Verlag 2007

Authors and Affiliations

  • Zhonglin Zhao
    • 1
  • Wei Lu
    • 2
  • Baoqing Dun
    • 3
  • Dan Jin
    • 4
  • Shuzhen Ping
    • 2
  • Wei Zhang
    • 2
  • Ming Chen
    • 2
  • Ming-Qun Xu
    • 5
  • Min Lin
    • 2
  1. 1.College of Biological SciencesChina Agricultural UniversityBeijingChina
  2. 2.Biotechnology Research InstituteChinese Academy of Agricultural SciencesBeijingChina
  3. 3.Institute of Crop ScienceChinese Academy of Agricultural SciencesBeijingChina
  4. 4.Biotechnology Research CenterSouthwest UniversityChongqingChina
  5. 5.New England BiolabsBeverlyUSA

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