Institute of Burn Research, Southwest Hospital, State Key Laboratory of Trauma, Burns and Combined Injury, The Third Military Medical University, Gaotanyan Street, Chongqing 400038, PR China.
Biomed Pharmacother. 2010 May;64(5):343-7. doi: 10.1016/j.biopha.2009.12.002. Epub 2009 Dec 30.
The trefoil factor family 2 (TFF2), a member of the trefoil factor family, plays a critical role in the defense and repair of gastrointestinal mucosa. However, its widespread application is hampered by difficulties in large-scale production of the recombinant protein suitable for clinical use. The aim of the present study was to produce hTFF2 by Escherichia coli expression system and explore its in vitro pharmacological characterization. hTFF2 gene encoding mature peptide was obtained by RT-PCR, and then inserted into the expression vector pET32a to construct the recombinant pET32a-hTFF2. After confirmation by gene sequencing, pET32a-hTFF2 was transformed into E. coli Origami B(DE3), and TrxA-hTFF2 fusion protein was expressed by conventional IPTG induction in a shake flask and analyzed with SDS-PAGE and Western-blot. Subsequently, TrxA-hTFF2 was isolated by Ni-NTA affinity chromatography, and ultrafiltration. Finally, we tested the effect of hTFF2 on cell migration in an in vitro restitution model and cell proliferation by MTT assay. The data revealed that the recombinant vector pET32a-hTFF2 was constructed successfully. TrxA-hTFF2 fusion protein was expressed to 246.5mg/L and its purity was above 95% after purification. SDS-PAGE and Western blot analyses showed that the fusion protein presented as a single band with a molecular weight of 32kDa. In vitro model of wounding demonstrated that hTFF2 enhanced migration activity by three folds. MTT assay exhibited a statistically significant dose-dependent growth-enhanced effect. Collectively, the results suggest that the recombinant hTFF2 was expressed in E. coli with high production, purity and biological activity.
三叶因子家族 2（TFF2）是三叶因子家族的成员，在胃肠道黏膜的防御和修复中发挥着关键作用。然而，由于难以大规模生产适合临床使用的重组蛋白，其广泛应用受到了阻碍。本研究旨在通过大肠杆菌表达系统生产 hTFF2，并探索其体外药理学特性。通过 RT-PCR 获得编码成熟肽的 hTFF2 基因，然后将其插入表达载体 pET32a 中，构建重组 pET32a-hTFF2。经基因测序确认后，将 pET32a-hTFF2 转化至大肠杆菌 Origami B（DE3）中，通过常规 IPTG 诱导在摇瓶中表达 TrxA-hTFF2 融合蛋白，并通过 SDS-PAGE 和 Western blot 进行分析。随后，通过 Ni-NTA 亲和层析和超滤法分离 TrxA-hTFF2。最后，我们在体外修复模型中测试了 hTFF2 对细胞迁移的影响，并通过 MTT assay 检测了细胞增殖的情况。结果表明，成功构建了重组载体 pET32a-hTFF2。表达的 TrxA-hTFF2 融合蛋白产量达到 246.5mg/L，纯度超过 95%，经纯化后。SDS-PAGE 和 Western blot 分析表明，融合蛋白呈现出一条分子量为 32kDa 的单带。体外划痕模型表明，hTFF2 可使迁移活性增强三倍。MTT assay 显示出具有统计学意义的剂量依赖性生长增强效应。综上所述，结果表明重组 hTFF2 在大肠杆菌中以高产、高纯度和高生物活性的形式表达。
