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Zoological Research
Kunming Institute of Zoology, Chinese Academy of Sciences
ISSN: 2095-8137
Vol. 32, No. 4, 2011, pp. 379-385
Bioline Code: zr11054
Full paper language: Chinese
Document type: Research Article
Document available free of charge

Zoological Research, Vol. 32, No. 4, 2011, pp. 379-385

 en Expression of Bm-TFF2 mutants in Escherichia coli check for this species in other resources and their cell migration-promoting activity
Yu, Guo-Yu; Xiang, Yang; Zhang, Hong-Yun; Jiang, Ping; Lee, Wen-Hui; Zhang, Yun & Zhang, Yong

Abstract

Bm-TFF2, a trefoil factor from the large-webbed bell toad ( Bombina maxima check for this species in other resources ), can stimulate cell migration and inhibit cell apoptosis. To study the structure-function relationship of Bm-TFF2, we constructed wild-type and mutated Bm-TFF2 plasmids and expressed recombinant proteins in E. coli. The wild-type Bm-TFF2 gene encoding mature peptide was obtained by RT-PCR, while the N-terminal, C-terminal and two arginine mutated Bm-TFF2 clones were constructed, and ligated into pET-32a(+) expression vectors. The fusion proteins were induced by IPTG at 37°C. The mutant Bm-TFF2 fusion proteins expressed mainly in the inclusion bodies. The mutant (TRX)/Bm-TFF2 could be purified by using Ni2+-chelating chromatography and reverse-phase HPLC from the inclusion body supernatant. The fusion proteins were analyzed by SDS-PAGE and Western blotting. The yield of mutant Bm-TFF2 fusion proteins of above 95% purity was about 20 mg/L. All three recombinant mutant proteins can promote the migration of AGS cells in a dose-dependent manner with no obvious activity difference.

Keywords
Bm-TFF2, Mutant Expression, Purification, Cell Migration

 
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