Wu Meiyu, Song Na, Cheng Jiliang, Zhao Yang, Chen Nazi, Ma Jisheng, Li Xiaokun, Jiang Chao, Wang Haijun
Department of Pathology, School of Basic Medical Sciences, Xinxiang Medical University, Xinxiang, Henan 453003, P.R. China.
Department of Molecular Biology and Biochemistry, School of Basic Medical Sciences, Xinxiang Medical University, Xinxiang, Henan 453003, P.R. China.
Mol Med Rep. 2017 Jul;16(1):447-452. doi: 10.3892/mmr.2017.6575. Epub 2017 May 11.
Fibroblast growth factor 17 (FGF17) is a novel member of the FGFs family, which is essential for cell development, tissue repair, tumor growth and invasion. The aim of the current study was to obtain a high expression level of recombinant human FGF17 (rhFGF17), including soluble proteins and inclusion bodies. An optimized rhFGF17 cDNA sequence was cloned into a pET3a vector, then the pET3a‑hFGF17 vector was transformed into BL21(DE3)pLysS Escherichia coli cells. Expression was induced by optimizing the conditions using isopropyl β‑D‑1‑thiogalactopyranoside (IPTG) and it was confirmed that a 24‑h exposure to 0.8 mM IPTG at 16˚C provided the optimal condition for soluble hFGF17. Furthermore, for the inclusion bodies, the optimal condition was a 4‑h exposure to 0.4 mM IPTG at 37˚C. Two forms of rhFGF17 protein were purified by heparin affinity and SP Sepharose Fast Flow chromatography. MTT assays demonstrated that the purified rhFGF17 exerted an important effect on the proliferative activity of NIH3T3 cells, although there was no significant difference when compared with standard rhFGF17. Thus, an optimal and economic expression system was created in the present study for rhFGF17 in E. coli. This expression strategy enables the preparation of sufficient and highly bioactive rhFGF17 for further investigation of underlying mechanisms.
成纤维细胞生长因子17（FGF17）是成纤维细胞生长因子（FGFs）家族的一个新成员，对细胞发育、组织修复、肿瘤生长和侵袭至关重要。本研究的目的是获得高表达水平的重组人FGF17（rhFGF17），包括可溶性蛋白和包涵体。将优化的rhFGF17 cDNA序列克隆到pET3a载体中，然后将pET3a-hFGF17载体转化到BL21(DE3)pLysS大肠杆菌细胞中。通过使用异丙基β-D-1-硫代半乳糖苷（IPTG）优化条件来诱导表达，结果证实，在16˚C下用0.8 mM IPTG处理24小时为可溶性hFGF17提供了最佳条件。此外，对于包涵体，最佳条件是在37˚C下用0.4 mM IPTG处理4小时。通过肝素亲和层析和SP Sepharose Fast Flow层析纯化了两种形式的rhFGF17蛋白。MTT分析表明，纯化的rhFGF17对NIH3T3细胞的增殖活性有重要影响，尽管与标准rhFGF17相比没有显著差异。因此，本研究在大肠杆菌中为rhFGF17建立了一个优化且经济的表达系统。这种表达策略能够制备足够的且具有高生物活性的rhFGF17，用于进一步研究其潜在机制。
