重组融合蛋白MBP-BSH在大肠杆菌中的表达及其纯化、功能鉴定.pdfVIP

198 2012, Vol. 33, No. 07 食品科学 ※生物工程 MBP-BSH 1 1 1,2 1, 黄 茜 ，黄 璐 ，潘道东 ，杨 瑶 * (1.南京师范大学 国家乳品加工技术研发分中心，江苏 南京 210097 ；2.宁波大学海洋学院，浙江 宁波 315211) 选择 IF2 融合标签，另选择含 SUMO 、GST 、NusA 和 MBP 融合标签的质粒构建表达载体。SDS 电泳结果显示，重组菌Escherichia Rosetta(DE3) (pLS932-BSH) 的诱导表达细胞提取液上清出现了明显的融合蛋白 MBP-BSH 条带，经诱导表达条件优化后，MBP-BSH 可溶性大幅度提高。用Ni-NTA Resin 和 Amylose Resin 分别 进行目标蛋白纯化，结果显示前者效果更好，并且 C 端融合 His-Tag 更有利于其与Ni 离子结合，MBP-BSH 蛋白 纯化量更大，杂带更少。茚三酮显色反应测定酶活力，结果显示纯化后的 MBP-BSH 的酶比活力为2.4282U/mg 。 融合蛋白 MBP-BSH ；表达纯化；功能鉴定；胆盐水解酶 Expression in Escherichia coli, Purification and Functional Characterization of Recombination Fusion Protein MBP-BSH 1 1 1,2 1, HUANG Qian ，HUANG Lu ，PAN Dao-dong ，YANG Yao * (1. Branch Center of National Dairy Products Processing Technology Development, Nanjing Normal University, Nanjing 210097, China ；2. School of Marine Sciences, Ningbo University, Ningbo 315211, China) Abstract ：The protein BSH, from Lactobacillu plantarum Y1, can form inclusion body when expressed in E. coli . The fusion tag IF2 can improve its solubility on the basis of our previous studies. In the present study, five fusion tags such as SUMO, GST, NusA, MBP and IF2 were used to improve the solubility of BSH. The results showed that an obvious protein band of MBP-BSH in the culture supernatant of recombinant E. coli Rosetta (DE3) (pLS932-BSH)was observed during the examination of SDS. However, after induction, its solubility was greatly improved. Meanwhile, Ni-NTA resin exhibited better purification