Purification of green fluorescent protein using a two-intein system

Appl Microbiol Biotechnol. 2008 Jan;77(5):1175-80. doi: 10.1007/s00253-007-1233-0. Epub 2007 Nov 1.


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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Chromatography, Affinity / methods*
  • Circular Dichroism
  • Fluorescence
  • Green Fluorescent Proteins / chemistry
  • Green Fluorescent Proteins / genetics
  • Green Fluorescent Proteins / isolation & purification*
  • Inteins*
  • Mutant Proteins / chemistry
  • Mutant Proteins / genetics
  • Mutant Proteins / isolation & purification*
  • Protein Binding / genetics
  • Protein Folding
  • Recombinant Fusion Proteins / chemistry
  • Recombinant Fusion Proteins / genetics
  • Recombinant Fusion Proteins / isolation & purification


  • Mutant Proteins
  • Recombinant Fusion Proteins
  • Green Fluorescent Proteins