重组rHSA-hFGF21融合蛋白在毕赤酵母中的表达纯化及活性分析  

Expression,purification and bioactivity analysis of a recombinant fusion protein rHSA-hFGF21 in Pichia pastoris

作　　者：黄甜甜 齐剑英 杨刚刚[1] 叶贤龙[1,2] HUANG Tiantian;QI Jianying;YANG Ganggang;YE Xianlong(College of Life Sciences,Henan Normal University,Xinxiang 453000,Henan,China;Ganjiang Traditional Chinese Medicine Innovation Center,Nanchang 330000,Jiangxi,China)

机构地区：[1]河南师范大学生命科学学院,河南新乡453000 [2]赣江中药创新中心,江西南昌330000

出　　处：《生物工程学报》2022年第9期3419-3432,共14页Chinese Journal of Biotechnology

基　　金：江西省自然科学基金(20212BAB206094);河南省青年托举项目(2021HYTP042);河南省重点研发与推广专项(科技攻关)(212102310242)。

摘　　要：人成纤维细胞生长因子21(human fibroblast growth factor 21,hFGF21)已成为改善血糖和脂质代谢的候选药物,但却存在半衰期短和易被体内代谢等缺点,导致靶组织利用率低,限制了其临床应用。本研究通过柔性连接肽(GGGGS)_(3)将hFGF21的N端连接至人血清白蛋白(human serum albumin,HSA)的C端,经过毕赤酵母密码子优化后,将重组基因片段rHSA-hFGF21连入pPIC9k和pPICZαA两种载体,并转化到X33、GS115和SMD1168三种不同酵母菌株中。通过加压筛选得到高表达的X33-pPIC9K-rHSA-hFGF21工程菌株,并优化摇瓶诱导条件如OD值、甲醇浓度和诱导时间进一步提高rHSA-hFGF21的表达效率。培养上清经中空纤维膜切向流技术粗分离、Blue亲和层析和Q离子交换层析纯化获得纯度约98.18%的rHSA-hFGF21蛋白。与hFGF21相比,rHSA-hFGF21具有更好的热稳定性和抗胰蛋白酶降解的能力,其血浆半衰期也延长了约27.6倍;中高浓度条件下rHSA-hFGF21刺激HepG2细胞摄取葡萄糖的能力优于hFGF21;在2型糖尿病动物中rHSA-hFGF21比hFGF21具有更好的长效降糖效果。本研究将为FGF21蛋白的大规模生产奠定基础。Human fibroblast growth factor 21(hFGF21) has become a candidate drug for regulating blood glucose and lipid metabolism. The poor stability and short half-life of hFGF21 resulted in low target tissue availability, which hampers its clinical application. In this study, the hFGF21 was fused with a recombinant human serum albumin(HSA), and the resulted fusion protein rHSA-hFGF21 was expressed in Pichia pastoris. After codon optimization, the recombinant gene fragment rHSA-hFGF21was inserted into two different vectors(pPIC9k and pPICZαA) and transformed into three different strains(X33, GS115 and SMD1168), respectively. We investigated the rHSA-hFGF21 expression levels in three different strains and screened an engineered strain X33-pPIC9K-rHSA-hFGF21 with the highest expression level. To improve the production efficiency of rHSA-hFGF21, we optimized the shake flask fermentation conditions, such as the OD value, methanol concentration and induction time. After purification by hollow fiber membrane separation, Blue affinity chromatography and Q ion exchange chromatography, the purity of the rHSA-hFGF21 protein obtained was 98.18%. Compared to hFGF21,the biostabilities of rHSA-hFGF21, including their resistance to temperature and trypsinization were significantly enhanced, and its plasma half-life was extended by about 27.6 times. Moreover, the fusion protein rHSA-hFGF21 at medium and high concentration showed a better ability to promote glucose uptake after 24 h of stimulation in vitro. In vivo animal studies showed that rHSA-hFGF21 exhibited a better long-term hypoglycemic effect than hFGF21 in type 2 diabetic mice. Our results demonstrated a small-scale production of rHSA-hFGF21, which is important for large-scale production and clinical application in the future.

关 键 词：成纤维细胞生长因子21(FGF21) 人血白蛋白(HSA) 融合蛋白 毕赤酵母 糖尿病 

分 类 号：Q78[生物学—分子生物学] R915[医药卫生—微生物与生化药学]