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

doi:10.7506/spkx1002-6630-201207042

食品科学 ›› 2012, Vol. 33 ›› Issue (7): 198-203.doi: 10.7506/spkx1002-6630-201207042

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

黄茜1，黄璐1，潘道东1,2，杨瑶1,*

1.南京师范大学国家乳品加工技术研发分中心 2.宁波大学海洋学院

出版日期:2012-04-15 发布日期:2012-04-20
基金资助:
江苏省高校自然科学基金项目(09KJB550003)

Expression in Escherichia coli, Purification and Functional Characterization of Recombination Fusion Protein MBP-BSH

HUANG Qian1，HUANG Lu1，PAN Dao-dong1,2，YANG Yao1,*

(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)

Online:2012-04-15 Published:2012-04-20

摘要/Abstract

摘要： 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-PAGE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。

关键词: 融合蛋白MBP-BSH, 表达纯化, 功能鉴定, 胆盐水解酶

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-PAGE. However, after induction, its solubility was greatly improved. Meanwhile, Ni-NTA resin exhibited better purification efficiency for MBP-BSH protein than Amylose resin. In addition, His-Tag linked at the C-terminal was favorable for the binding of nickel ions and as a result, MBP-BSH containing less impurities was obtained in a higher yield. The enzymatic activity of purified BSH was 2.4282 U/mg (115.14 AU/mg) as determined by ninhydrin color reaction.

Key words: fusion protein MBP-BSH, expression and purification, functional characterization, bile salt hydrolase

中图分类号:

TS201.3

黄茜，黄璐，潘道东，杨瑶. 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[J]. 食品科学, 2012, 33(7): 198-203.

HUANGQian，HUANGLu，PANDao-dong，YANGYao. Expression in Escherichia coli, Purification and Functional Characterization of Recombination Fusion Protein MBP-BSH[J]. FOOD SCIENCE, 2012, 33(7): 198-203.

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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

Expression in Escherichia coli, Purification and Functional Characterization of Recombination Fusion Protein MBP-BSH

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