提高成纤维细胞生长因子-21产率和纯度

发布时间：2018-09-01 17:04

【摘要】：成纤维细胞生长因子-21(FGF-21)作为近期发现的新型代谢调节因子,因其具有独立于胰岛素调节糖脂代谢、增加胰岛素敏感性等作用,有望成为治疗糖尿病的新型药物。包涵体形式表达外源蛋白表达量及纯度高,但是以pET载体表达时,FGF-21以包涵体形式表达,且复性率及产率低,蛋白活性降低[1]。针对这一瓶颈问题,用SUMO载体首次以包涵体形式表达带有SUMO标签的hFGF-21,通过优化培养条件,并应用中空纤维柱膜过滤技术对菌体进行富集,对包涵体进行洗涤、变性及复性,经过亲和层析、凝胶过滤层析的纯化方法,得到了成熟的hFGF-21,在保证蛋白活性的同时增加了蛋白的产量及纯度。通过检测HepG2细胞葡萄糖吸收及2型糖尿病db/db小鼠短期及长期血糖变化鉴定其降糖生物学活性。结果表明,以包涵体形式表达hFGF-21(ihFGF-21)的表达量是可溶形式表达的hFGF-21(shFGF-21)的3倍,最终ihFGF-21的收率为20 mg/L,而shFGF-21的收率仅为6 mg/L。ihFGF-21的纯度可达到95%以上,而shFGF-21仅能达到90%左右;在细胞水平和动物水平上两者的降糖生物学活性一致。在保证hFGF-21生物学活性的前提下,与传统包涵体途径提取目的蛋白的方法相比,应用中空纤维柱膜过滤技术使hFGF-21的生产周期缩短了约1/3左右。综上所述,此法为FGF-21中试及工业化生产提供了高效、经济的策略。
[Abstract]:Fibroblast growth factor-21 (FGF-21), as a newly discovered metabolic regulator, is expected to be a new drug for diabetes mellitus because of its independent effects on insulin-mediated glucose and lipid metabolism and increased insulin sensitivity. To solve this bottleneck problem, the inclusion body of hFGF-21 with SUMO tag was expressed in the form of inclusion body for the first time. The culture conditions were optimized, and the cell was enriched by hollow fiber membrane filtration technology, and the inclusion body was washed, denatured and renatured. The mature hFGF-21 was obtained by superaffinity chromatography and gel filtration chromatography. The yield and purity of the protein were increased while the activity of the protein was guaranteed. The hypoglycemic biological activity was evaluated by detecting glucose uptake in HepG2 cells and short-term and long-term blood glucose changes in type 2 diabetic db/db mice. The final yield of ihFGF-21 was 20 mg/L, while the purity of shFGF-21 was only 6 mg/L. The purity of shFGF-21 was above 95%, while that of shFGF-21 was only about 90% at the cellular and animal levels. On the premise of biological activity of F-21, the production cycle of hFGF-21 was shortened about one third by using hollow fiber column membrane filtration compared with the traditional inclusion body method.
【作者单位】：东北农业大学生命科学学院生物制药教研室;
【基金】：黑龙江省发展和改革委员会重大前期项目(No.黑发改项目[2011]1570) 黑龙江省博士后科研启动基金项目(No.LBH-Q09162) 黑龙江省教育厅科学技术研究项目(No.12521z004) 国家自然科学基金(No.J1210069/J0106)资助~~
【分类号】：R318.0

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