Purification of green fluorescent protein using a two-intein system

文献类型： 外文期刊

第一作者： Lu, Wei

作者： Lu, Wei;Ping, Shuzhen;Zhang, Wei;Chen, Ming;Lin, Min;Zhao, Zhonglin;Dun, Baoqing;Jin, Dan;Xu, Ming-Qun

作者机构：

关键词： intein;protein splicing;green fluorescent protein;protein purification;EXPRESSED PROTEINS;ESCHERICHIA-COLI;INTEIN;LIGATION;CYCLIZATION;STEP

期刊名称：APPLIED MICROBIOLOGY AND BIOTECHNOLOGY （ 影响因子：4.813； 五年影响因子：4.697 ）

ISSN：

年卷期：

页码：

收录情况： SCI

摘要： 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.

分类号： Q93

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