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以C端为甘氨酸的修饰型人降钙素 (mhCT Gly)的融合蛋白为底物和利用重组酰胺化酶 ,研究建立基因工程表达产物的体外酰胺化加工系统。首先 ,人工合成mhCT Gly基因 ,并构建其融合表达质粒 pGEXCT ,在大肠杆菌中获得了高效表达并通过亲和层析分离纯化获得谷胱甘肽S 转移酶 (GST)融合蛋白 (GST mhCT Gly)。同时 ,从稳定表达大鼠酰胺化酶的CHO细胞株中制备了重组酰胺化酶。然后 ,利用此重组酰胺化酶对GST mhCT Gly进行体外酰胺化加工研究。特异性抗体免疫结合实验和质谱鉴定结果表明 ,融合表达蛋白GST mhCT Gly已被重组酰胺化酶酰胺化加工修饰。
The fusion protein of modified human calcitonin (mhCT Gly) with C-terminal glycine as substrate and recombinant amidase were used to study the establishment of an in vitro amidation processing system of genetically engineered expression product. Firstly, the mhCT Gly gene was synthesized and its fusion expression plasmid pGEXCT was constructed. The recombinant plasmid pGEXCT was highly expressed in E. coli and purified by affinity chromatography to obtain the GST fusion protein (GST mhCT Gly) . In the meantime, recombinant amidase was prepared from a CHO cell line stably expressing rat amidase. Then, GST mhCT Gly was subjected to in vitro amidation studies using this recombinant amidase. Specific antibody immunobinding assay and mass spectrometry showed that the fusion protein GST mhCT Gly was modified by amidation of recombinant amidase.