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- Title
Purification of green fluorescent protein using a two-intein system.
- Authors
Zhonglin Zhao; Wei Lu; Baoqing Dun; Dan Jin; Shuzhen Ping; Wei Zhang; Ming Chen; Ming-Qun Xu; Min Lin
- 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.
- Publication
Applied Microbiology & Biotechnology, 2008, Vol 77, Issue 5, p1175
- ISSN
0175-7598
- Publication type
Academic Journal
- DOI
10.1007/s00253-007-1233-0