富含二硫键的膜蛋白p185胞外区在大肠杆菌中的可溶性表达和性质鉴定  

Soluble Expression and Characterization of Disulfide Bond-rich Subdomains of Membrane Protein p185 in Escherichia coli

作　　者：李良维[1] 刘海波[1] 胡思怡[1] 梁顿[1] 程联胜[1] 刘兢[1] 

机构地区：[1]中国科学技术大学生命科学学院细胞免疫学实验室,合肥230027

出　　处：《生物工程学报》2005年第4期590-596,共7页Chinese Journal of Biotechnology

基　　金：国家高技术研究与发展计划项目(No.2001AA215381);国家自然科学基金资助(No.30400078);高等学校博士学科点专项科研基金资助(No.20020358048)~~

摘　　要：Her2/c_erbB_2基因(其产物为膜蛋白p185)是表皮生长因子受体(EGFR)基因家族的一员,在约30%的乳腺癌中发现了其过量表达。为了鉴定抗p185单克隆抗体的抗原表位并进一步研究它们的相互作用,采用PCR的方法从含Her2/c_erbB_2基因的pBabe/erbB_2质粒中扩增了p185胞外区的富含二硫键的第一、二结构域和第四个结构域。产物克隆到pGEX/4T_1载体后,转化大肠杆菌OrigamiB(DE3)pLysS菌株,用低浓度IPTG进行低温过夜诱导后将菌体压力破碎,SDS_PAGE检测表达上清,得到了可溶性表达的融合有GST的目的蛋白。经ELISA、Westernblot等方法鉴定,可溶性表达产物具有完全的抗体结合活性,且当用凝血酶把GST切掉后该活性仍然保留。p185胞外区融合蛋白的成功表达将为二硫键富含类蛋白的表达提供参考;并为将来具有肿瘤细胞生长抑制活性的抗p185单克隆抗体的抗原表位鉴定,以及为EGFR家族受体的结构和功能关系的研究打下基础。Transmembrane protein p185 (the product of Her2/c-erbB-2 gene) is a member of the epidermal growth factor receptor (EGFR) family. Its overexpression was found in about 30% of breast cancer. It is essential to obtain soluble extracellular domain (ECD) of p185, especially disulfide bond rich domains, for identifying the epitopes of anti-p185 antibodies and researching the interrelationship between the antigen and antibody. The disulfide bond rich domain Ⅰ-Ⅱ and domain IV of p185 ECD were amplified from plasmid pBabe/erbB-2 by PCR respectively. These two fragments were inserted into pGEX/4T-1 vector, transfected into E.coli Origami B(DE3)pLysS and expressed inductively by low concentration of IPTG and low temperature overnight. After the pressure lysis of cells, the supernatants were analyzed by SDS-PAGE and the result demonstrated that this GST-fusion protein was expressed solubly in the amount of 10～15mg/L. By the ELISA, Western blot and other immunological assays, the fusion proteins and their GST cut-off derivates both showed binding activities with several anti-p185 antibodies respectively. These results indicated that it was a feasible and effectual method to express disulfide bond rich domain Ⅰ-Ⅱ and domain Ⅳ of p185 ECD and this method may also be used to express other disulfide bond rich proteins.

关 键 词：p185胞外区 融合蛋白 谷胱甘肽S转移酶 

分 类 号：R73-3[医药卫生—肿瘤]