融合蛋白GGHN端延长类似物的克隆表达、纯化及活性研究

Expression,Purification and Bioactivity of Fusion Protein GGH Analogue with N-terminal Extension

GGH是人胰高血糖素样肽-1与人血清白蛋白融合蛋白(GLP-1A2G)2-HSA的简称,为研究N端添加丙氨酸的修饰对其活性的影响,通过基因工程方法成功构建表达该类似物的工程菌,并纯化得到终产物,完成了初步体外活性检测。首先运用PCR技术扩增出融合蛋白GGH类似物的基因片段,以pPIC9K为表达质粒构建了重组表达质粒,将双酶切和测序验证正确的重组质粒电转至分泌型菌株毕赤酵母GS115,然后经过MD平板筛选获得转化子,利用甲醇诱导表达72 h,SDS-PAGE检测上清产物与未修饰的GGH对比,得到阳性转化子。融合蛋白经过发酵液8 000 r/min离心10 min,0.45μm的微孔滤膜微滤,采用截留分子量为10 kD的超滤膜超滤20倍,再依次经过Blue Sepharose 6 Fast Flow、Phenyl Sepharose Fast Flow、Q Sepharose Fast Flow、Sephadex G-25层析柱。在RINm5f细胞上测cAMP的生成,通过与阳性对照的标准曲线对比发现,A-GGH活性比GGH提高10倍,表现出较好的体外活性。

GGH is a fusion protein of two human glucagon-like peptide-1 mutant and human serum albumin.In order to check the influence of the extension of an alanine to the N-terminal of GGH,we constructed recombinant plasmid pPIC9K/A-GGH capable of expressing the analogue in the methylotrophic yeastP.pastorisGS115.Recombinant strainP.pastorisGS115 was screened through MD plates.After 72 h of inducing by 2% methanol at 30℃,the fusion protein was purified from fermentation broth by centrifugation,ultrafiltration concentration,affinity absorption chromatography,ion exchange chromatography and gel filtration.Activity test result suggested that A-GGH showed better activity in vitroand that its cAMP level was significantly increased by 10-fold compared to GGH without N-terminal extension.