College of Life Science, Henan Normal University, No. 46 construction road, 453007 Xinxiang, Henan Province, China.
College of Life Science, Northeast Agricultural University, No. 59 Mucai Street, 150030 Harbin, Heilongjiang Province, China.
Int J Biol Macromol. 2019 Aug 15;135:362-372. doi: 10.1016/j.ijbiomac.2019.05.167. Epub 2019 May 24.
Fibroblast growth factor 21 (FGF-21) is an important regulator in glycolipid metabolism that is a promising drug candidate for treatment of diabetes and obesity. However, the productivity of recombinant hFGF-21 (rhFGF-21) in Escherichia coli (E. coli) is relatively low, which limits its clinical application. To meet the clinical demand and control the production cost, rhFGF-21 proteins were expressed in inclusion bodies (IBs) form in Rosetta (DE3) by high cell density fermentation in 50-L scale. Hollow fiber membrane filtration technology was used to enrich the bacteria, wash, denature and refold the IBs in the current report. The renatured proteins were purified by two-step affinity chromatography. Authenticity of the purified rhFGF-21 was confirmed by the N-and C-terminal sequence, disulfide bond composition and molecular weight analyses. Results showed that the average target protein and recovery of rhFGF-21 expressed in IBs form of three batches were more than those of the soluble form. Both the rhFGF-21 proteins from the two forms showed equal potency in improving the glucose uptake in HepG2 cells and anti-diabetic effect in db/db mice. In this study, an efficient method for preparation of FGF-21 was established. This novel process provides an important technical basis for the large-scale production of rhFGF-21.
成纤维细胞生长因子 21（FGF-21）是糖脂代谢的重要调节剂，是治疗糖尿病和肥胖症的有前途的药物候选物。然而，大肠杆菌（E. coli）中重组 hFGF-21（rhFGF-21）的产率相对较低，限制了其临床应用。为了满足临床需求并控制生产成本，rhFGF-21 蛋白在罗氏（DE3）中以包涵体（IB）形式通过 50-L 规模的高密度发酵进行表达。中空纤维膜过滤技术用于富集细菌，在当前报告中对 IB 进行洗涤、变性和重折叠。两步亲和层析法对复性蛋白进行纯化。通过 N 和 C 末端序列、二硫键组成和分子量分析证实了纯化的 rhFGF-21 的真实性。结果表明，三批 IB 形式表达的平均目标蛋白和 rhFGF-21 的回收率均高于可溶性形式。两种形式的 rhFGF-21 蛋白在改善 HepG2 细胞葡萄糖摄取和 db/db 小鼠抗糖尿病作用方面均具有同等效力。在这项研究中，建立了一种有效的 FGF-21 制备方法。该新工艺为 rhFGF-21 的大规模生产提供了重要的技术基础。
