title: Bacterial expression and purification of biologically active human TFF2.
abstract: Human trefoil factor 2 (hTFF2) is considered as one of the most important initiators of mucosal healing in the gastrointestinal tract by promoting cell migration and suppressing apoptosis. However, it is hard to obtain hTFF2 from human tissue and many recombinant hTFF2 produced in vitro exist as fusion proteins. The purpose of the present study was to produce native hTFF2 while maintaining its biological activities. The open reading frame of hTFF2 was inserted into a pET-32a(+) expression vector, and hTFF2-TRX fusion protein was successfully expressed in Escherichia coli and purified by Nickel-nitrilotriacetic acid affinity chromatography and reverse-phase HPLC steps. The recombinant fusion protein (purity>95%) was cleaved by Factor Xa at 23 Degrees Celsius to release hTFF2. After removal of Factor Xa and undigested fusion proteins, hTFF2 was purified and identified by SDS-PAGE and Western blotting. The yield of recombinant hTFF2 was about 5 mg/L. The recombinant hTFF2 could promote IEC-6 cells migration and in vitro wound healing via the activation of ERK1/2. Recombinant hTFF2 could also inhibit apoptosis of HCT-116 cells induced by 50 μmol/L ceramide. In summary, our results showed that the recombinant hTFF2 was expressed in E. coli and successfully purified after cleavage with the fusion partner with high yield while maintaining its biological activities. Recombinant hTFF2 might be useful for investigating the molecular mechanism of hTFF2 and development of hTFF2-related drugs.
authors.affiliation: Chinese Academy of Sciences, Kunming Institute of Zoology, Kunming, Yunnan, China.
authors.forename: Yong-Hui
authors.initials: YH
authors.lastname: Zhuang
authors.forename: Si-Man
authors.initials: SM
authors.lastname: Li
authors.forename: Guo-Yu
authors.initials: GY
authors.lastname: Yu
authors.forename: Yong
authors.initials: Y
authors.lastname: Zhang
authors.forename: Yang
authors.initials: Y
authors.lastname: Xiang
authors.forename: Hao
authors.initials: H
authors.lastname: Zou
authors.forename: Wen-Hui
authors.initials: WH
authors.lastname: Lee
mesh_terms: D015153:Blotting, Western; D002846:Chromatography, Affinity; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D010455:Peptides / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism*; D000071164:Trefoil Factor-2
chemical_list: D010455:Peptides; C000604007:TFF2 protein, human; D000071164:Trefoil Factor-2