Applied Microbiology and Biotechnology

, Volume 77, Issue 5, pp 1175–1180

Purification of green fluorescent protein using a two-intein system

Authors

  • Zhonglin Zhao
    • College of Biological SciencesChina Agricultural University
  • Wei Lu
    • Biotechnology Research InstituteChinese Academy of Agricultural Sciences
  • Baoqing Dun
    • Institute of Crop ScienceChinese Academy of Agricultural Sciences
  • Dan Jin
    • Biotechnology Research CenterSouthwest University
  • Shuzhen Ping
    • Biotechnology Research InstituteChinese Academy of Agricultural Sciences
  • Wei Zhang
    • Biotechnology Research InstituteChinese Academy of Agricultural Sciences
  • Ming Chen
    • Biotechnology Research InstituteChinese Academy of Agricultural Sciences
  • Ming-Qun Xu
    • New England Biolabs
    • Biotechnology Research InstituteChinese Academy of Agricultural Sciences
Methods

DOI: 10.1007/s00253-007-1233-0

Cite this article as:
Zhao, Z., Lu, W., Dun, B. et al. Appl Microbiol Biotechnol (2008) 77: 1175. doi:10.1007/s00253-007-1233-0

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

InteinProtein splicingGreen fluorescent proteinProtein purification

Copyright information

© Springer-Verlag 2007