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.
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This work was supported by Foundation of 863 Project of China.
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Zhao, Z., Lu, W., Dun, B. et al. Purification of green fluorescent protein using a two-intein system. Appl Microbiol Biotechnol 77, 1175–1180 (2008). https://doi.org/10.1007/s00253-007-1233-0
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DOI: https://doi.org/10.1007/s00253-007-1233-0