title: [Expression of Bm-TFF2 mutants in Escherichia coli and their cell migration-promoting activity].
abstract: Bm-TFF2, a trefoil factor from the large-webbed bell toad (Bombina maxima), 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 Degrees Celsius. The mutant Bm-TFF2 fusion proteins expressed mainly in the inclusion bodies. The mutant (TRX)/Bm-TFF2 could be purified by using Ni(2+)-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.
authors.affiliation: Key Laboratory of Animal Models and Human Disease Mechanisms, Kunming Institute of Zoology, the Chinese Academy of Sciences, Kunming Yunnan 650223, China.
authors.forename: Guo-Yu
authors.initials: GY
authors.lastname: Yu
authors.forename: Yang
authors.initials: Y
authors.lastname: Xiang
authors.forename: Hong-Yun
authors.initials: HY
authors.lastname: Zhang
authors.forename: Ping
authors.initials: P
authors.lastname: Jiang
authors.forename: Wen-Hui
authors.initials: WH
authors.lastname: Lee
authors.forename: Yun
authors.initials: Y
authors.lastname: Zhang
authors.forename: Yong
authors.initials: Y
authors.lastname: Zhang
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D001001:Anura / Q000235:genetics*; D045744:Cell Line, Tumor; D002465:Cell Movement / Q000187:drug effects*; D004926:Escherichia coli / Q000235:genetics* / Q000378:metabolism; D015870:Gene Expression; D006801:Humans; D008969:Molecular Sequence Data; D009154:Mutation; D010455:Peptides / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism / Q000494:pharmacology*; D011994:Recombinant Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism / Q000494:pharmacology; D000071164:Trefoil Factor-2
chemical_list: D010455:Peptides; D011994:Recombinant Proteins; C000604007:TFF2 protein, human; D000071164:Trefoil Factor-2