提高成纤维细胞生长因子-21产率和纯度

Improvement of yield and purity of human fibroblast growth factor-21

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) is a recently discovered metabolic regulation factor, regulating glucose and lipid metabolism and increasing insulin sensitivity. FGF-21 is expected to be a potential anti-diabetic drug. Expression of FGF-21 as inclusion bodies has advantages for high yield and purity, but the bioactivity of the protein is almost totally lost after denature and renature. That is why FGF-21 is currently expressed in soluble form. As a result, the yield is considerably low. In this study, we used SUMO vector to express SUMO-human FGF-21 (SUMO-hFGF-21) in form of inclusion body. We optimized the culture conditions to increase the yield of the bioactive human fibroblast growth factor-21. We applied the hollow fiber membrane filtration column to enrich the bacteria, wash, denature and renature inclusion bodies. After affinity and gel filtration chromatography, we examined the hypoglycemic activity of FGF-21 by the glucose uptake assay in HepG2 cells. We also detected the blood glucose concentration of type 2 diabetic db/db model mice after short or long-term treatment. The results show that the yield of ihFGF-21 was 4 times higher than that of shFGF-21. The yield was 20 mg/L for ihFGF-21 vs. 6 mg/L for shFGF-21. The purity of ihFGF-21 was above 95%, while shFGF-21 was 90%. Compared with the traditional method of extracting inclusion bodies, the production cycle was about three times shortened by application of hollow fiber membrane filtration column technology, but the bioactivity did not significantly differ. This method provides an efficient and cost-effective strategy to the pilot and industrial production of hFGF-21.