棉铃虫组织蛋白酶B酶原在大肠杆菌中的表达及纯化.pdf

农业生物技术学报 Journal of Agricultural Biotechnology 2004,12 (2): 162~ 166 ·研究论文· 棉铃虫组织蛋白酶B 酶原在大肠杆菌中的表达及纯化* 欣军 邵红莲 邵丁丁 赵小凡** 王金星 (山东大学生命科学院生化与分子生物学研究所, 济南250100) 摘要: 用合成的基因特异性引物通过RT-PCR 扩增，获 棉铃虫( )组织蛋白酶B 基因（ ），将 因克隆到pGEX-4T-1 载体，在大肠杆菌( )DH5 内进行扩增，经过PCR 筛选获得阳性克隆并提取质粒和测序 ，再 验证后 转化大肠杆菌BL21 进行诱导表达。表达产物为组织蛋白酶B- 谷胱甘肽S 转移酶的融合蛋白,分子量在63 kD 左 右。表达产物形成了包涵体，经过对包涵体变性和复性处理， 到了可溶性的融合蛋白，可被Glutathione Sepharose 4B 柱亲和 ，经 。 纯化，用凝血酶裂解融合蛋白后 SDS分离到37 kD 左右的棉铃虫组织蛋白酶B 酶原 N- 末端氨 酸测序鉴定表达 产物为棉铃虫组织蛋白酶B 。 关键词: 棉铃虫；组织蛋白酶B ；重组表达；包涵体；复性 Expression and Purification of Procathepsin B from in DU Xin-Jun SHAO Hong-Lian SHAO Ding-Ding ZHAO Xiao-Fan** WANG Jin-Xing (Biochemistry and molecular biology institute, School of Life Sciences, Shandong University, Jinan 250 100,China) The cathepsin B cDNA from was cloned by RT-PCR using gene specific primers. After ligated the cDNA into pGEX-4T-1 vector, the recombinant plasmid was amplified in competent DH5 . Positive clones were selected and the recombinant plasmid was isolated from DH5 and identified by sequencing. A 63 kD fusion protein including the procathepsin B and glutathions-transferase (GST) was expressed in BL21 by induction of isopropylthio-茁-D-galactoside(IPTG). However, the expressed product formed complete inclusion bodies. After denature with 8 mol/L urea and refolding by dialysis in Tris-HCl buffer, a