人GLP-1-IgG Fc融合蛋白在毕赤酵母中的高效表达  

Expression of Human GLP-1-IgG Fc Fusion Protein in Pichia pastoris

作　　者：王圣钧[1] 郁慧丽[1] 翟琳[1] 王倩[2] 梁泉峰[1] 祁庆生[1,2] 

机构地区：[1]山东大学生命科学学院微生物技术国家重点实验室,山东济南250100 [2]山东大学国家糖工程技术研究中心,山东济南250100

出　　处：《生物技术》2011年第4期29-33,共5页Biotechnology

基　　金：山东省自然科学基金项目("结合菌种改造与蛋白质工程的胰高血糖素样肽-1(GLP-1)表达研究";ZR2010CM029)资助

摘　　要：目的:构建GLP-1-IgG Fc融合蛋白分子并在毕赤酵母中实现高效表达。方法:使用蛋白质工程技术改造GLP-1,去除其蛋白酶降解位点,然后利用重叠延伸PCR方法得到改造后的GLP-1与人IgG-Fc片断的嵌合体基因并将其插入pPIC9K载体中。以重组载体转化巴斯德毕赤酵母菌中进行表达。采用SDS-PAGE和Western Blot方法检测重组蛋白的表达。结果:成功的构建了GLP-1-IgG Fc嵌合体基因并使其在重组毕赤酵母中高效分泌表达。在25℃条件下,摇瓶培养添加0.5%甲醇诱导72h后融合蛋白的表达量最大,为5mg/L。SDS-PAGE和Western-Blot结果表明表达产物为GLP-1-IgG Fc融合蛋白。结论:获得了高效表达GLP-1-IgG Fc融合蛋白的毕赤酵母菌株,为GLP-1-IgG Fc的活性和半衰期测定及下一步的开发奠定了基础,并为在毕赤酵母菌中表达其他Fc融合蛋白和抗体提供了参考。Objective：Construct and high-effectively express the fusion protein GLP-1-IgG Fc in the Pichia pastoris.Method：Modified the sequence of GLP-1 by eliminating its peptide enzymes sites.The chimeric gene GLP-1-IgG Fc achieved through gene splicing by overlap extension（SOE） method was cloned into pPIC9K and transformed into Pichia pastoris.The fusion protein was expressed in Pichia pastoris and analyzed using SDS-PAGE and Western Blot.Result：The chimeric gene GLP-1-IgG Fc and yeast expression vector pPIC9k/GLP-1-IgG Fc were constructed.The fusion protein GLP-1-IgG Fc was successfully expressed in the Pichia pastoris and the expression level of fusion protein in 1 L flask reached 5 mg/L after 72h growth.The results of SDS-PAGE and Western-Blot showed the expressed protein was the GLP-1-IgG Fc fusion protein.Conclusion：The human GLP-1-IgG Fc fusion protein was expressed successfully in Pichia pastoris,and it could be the foundation for the GLP-1-IgG Fc activity and half-life determination.

关 键 词：胰高血糖素样肽-1 FC融合蛋白 巴斯德毕赤酵母菌 高效表达 

分 类 号：Q785[生物学—分子生物学] Q786