hIAPP与麦芽糖结合蛋白的融合表达、纯化和鉴定  

Expression,Purification and Identification of hIAPP Fused with Maltose Binding Protein

作　　者：尚金召 王雪芹 熊聿力 路福平[1] 刘夫锋 SHANG Jinzhao;WANG Xueqin;XIONG Yuli;LU Fuping;LIU Fufeng(State Key Laboratory of Food Nutrition and Safety,Key Laboratory of Industrial Fermentation Microbiology,Ministry of Education,National Engineering Laboratory for Industrial Enzymes,College of Biotechnology,Tianjin University of Science&Technology,Tianjin 300457,China)

机构地区：[1]食品营养与安全国家重点实验室工业发酵微生物教育部重点实验室工业酶国家工程实验室天津科技大学生物工程学院

出　　处：《天津科技大学学报》2019年第6期25-30,41,共7页Journal of Tianjin University of Science & Technology

基　　金：国家自然科学基金资助项目(21576199);天津市科技支撑计划资助项目(18JCZDJC33000)

摘　　要：基于人胰岛淀粉样多肽(hIAPP)的氨基酸序列,按照大肠杆菌(E.coli)密码子偏好性合成基因,并克隆到pMAL-c2x载体中,与MBP蛋白malE基因融合表达,获得重组表达质粒pMAL-hIAPP.将质粒pMAL-hIAPP转化到表达宿主E.coli BL21(DE3)中,利用异丙基硫代半乳糖苷(IPTG)诱导融合蛋白MBP-hIAPP表达.SDS-PAGE电泳显示融合蛋白MBP-hIAPP的相对分子质量约为4.9×104.利用重组型烟草蚀纹病毒的半胱氨酸蛋白酶(rTEV)酶切MBP-hIAPP,并采用凝胶过滤层析的方法分离出高纯度的hIAPP,利用MALDI-TOF质谱进行了验证.Based on the amino acid sequence of human islet amyloid polypeptide(hIAPP),the corresponding genes were synthesized according to E.coli codon bias.The hIAPP gene was cloned into pMAL-c2x vector,expressed with MBP protein and the expression vector pMAL-hIAPP was obtained.pMAL-hIAPP was then transformed into the expression host E.coli BL21(DE3).Subsequently,the engineering bacteria BL21-pMAL-hIAPP was induced with isopropyl thiogalactoside(IPTG).SDS-PAGE electrophoresis showed that the molecular weight of the fusion protein MBP-hIAPP was approximately 4.9×104.MBP-hIAPP was digested with recombinant tobacco etch virus cysteine protease(rTEV),and high purity hIAPP was isolated by using gel filtration chromatography,which was further confirmed by MALDI-TOF MS spectra.

关 键 词：人胰岛淀粉样多肽 麦芽糖结合蛋白 融合表达 纯化 鉴定 

分 类 号：Q816[生物学—生物工程]