定点突变FGF21提高表达量研究

Enhancement of FGF21 expression by site-directed mutagenesis

FGF21是一类具有重要糖脂代谢调节功能因子,是治疗2型糖尿病候选药物。但其在大肠杆菌中表达量低且主要以包涵体形式表达,为此根据FGF21与FGF家族同源序列差异,对FGF21进行定点突变以提高其表达量。以pSUMO-FGF21表达载体为模板,在突变位点两端设计引物,采用定点突变方法,将FGF21141位甘氨酸(G)突变为苯丙氨酸(F),构建突变FGF21表达载体pSUMO-mutFGF21;在大肠杆菌中进行表达,纯化mutFGF21蛋白。利用SDS-PAGE和Western blot鉴定分析突变蛋白,HepG2细胞糖吸收试验检测突变蛋白活性。结果成功对pSUMO-hFGF21进行突变,突变蛋白在大肠杆菌中成功表达,经SDS-PAGE分析,主要以可溶形式表达;与野生型FGF21(hFGF21)相比,表达量提高50%。Western blot表明mutFGF21可与FGF21抗体特异性反应。糖吸收试验显示mutFGF21具有与hFGF21一样生物活性并存在剂量依赖性,相比hFGF21,mutFGF21活性略有提高,说明定点突变FGF21(141位G突变为F)可显著提高FGF21表达量。

FGF21, an important metabolic regulator of both glucose and lipid, is believed to have great potential in treating metabolic diseases, especially diabetes, but FGF21 expression in E.coli is rather low and mainly in the form of inclusion body hampering the drug development of the molecule. To overcome the problem, a conserved amino acid of FGF21 was mutated by site-directed mutagenesis expecting an increase in production. A pair of complementary primer was designed around the mutation site, using the expression vector pSUMO-FGF21 as the template, by PCR the 141th G of FGF21 gene was mutated into F. The mutated FGF21 protein was expressed in Rosetta （DE3） and the purified protein was analyzed by SDS-PAGE and Westem blot. The biological activity of the mutFGF21 was tested on HepG2 cells and compared with that of the wild type FGF21 （hFGF21）. The results showed the pSUMO-mutFGF21 vector was successfully constructed and expressed in Rosetta mostly in soluble form. The mutFGF21 protein has a 1.5 fold higher production than that of hFGF21. The biological activity results indicate the mutFGF21 has the same glucose uptake efficiency in HepG2 cells as hFGF21 in a dose-dependent manner. Compared withhFGF21, mutFGF21 has a better performance on production.