A new tagged-TEV protease Construction, optimisation of production, purification and test activity

被引：26

作者：

Miladi, Baligh ^{[1
]}

Bouallagui, Hassib ^{[2
]}

Dridi, Cyrine ^{[1
]}

El Marjou, Ahmed ^{[3
]}

Boeuf, Guilhem ^{[1
]}

Di Martino, Patrick ^{[4
]}

Dufour, Florence ^{[1
]}

Elm'Selmi, Abdellatif ^{[1
]}

机构：

[1] Ecole Biol Ind, Mol Biol Lab, F-95094 Cergy, France

[2] Natl Inst Appl Sci & Technol, Lab Microbial Ecol & Technol, Tunis 1080, Tunisia

[3] Inst Curie, CNRS, UMR144, Lab Recombinant Prot, F-75231 Paris, France

[4] Univ Cergy, ERRMECe Lab EA1391, Pontoise, France

来源：

PROTEIN EXPRESSION AND PURIFICATION | 2011年 / 75卷 / 01期

关键词：

Streptag II-TEV; Solubility; High level of production; Affinity chromatography; ETCH VIRUS PROTEASE; ONE-STEP PURIFICATION; STREP-TAG; ESCHERICHIA-COLI; AFFINITY TAGS; RECOMBINANT PROTEINS; FUSION PROTEIN; II PEPTIDE; EXPRESSION; STREPTAVIDIN;

D O I：

10.1016/j.pep.2010.08.012

中图分类号：

Q5 [生物化学];

学科分类号：

071010 ; 081704 ;

摘要：

The Tobacco Etch Virus (TEV) protease is frequently used in the cleavage of recombinant fusion proteins because of its efficiency and high specificity In this work we present a new recombinant form of TEV termed Streptag II-TEV for high-level production and purification of TEV protease from Escherichia colt and compare It to the hexahistidine (6xHis) tagged version of TEV The effects of varying the host strain the bacterial induction temperature (25 30 and 37 degrees C) and the IPTG inducer concentration on production and solubility of the two recombinant TEV proteases have been examined Optimal Streptag II-TEV protein expression were obtained in the E colt KRX strain under an induction temperature of 25 degrees C in the presence of IPTG at 0 5 mM In these conditions soluble Streptag II-TEV and 6xHis-TEV proteases accounted for about 25% and 18% of total soluble proteins respectively About 70% of Streptag II-TEV and 60% of 6xHis-TEV were detected in the supernatant Streptag II-TEV protease purifies to near homogeneity (approximately 99%) via a simple single step Strep-Tactin chromatography purification protocol based on the presence of Streptag II The higher production of Streptag II-TEV coupled to its purification and cleavage efficiencies make it an attractive alternate to 6xHis-TEV (C) 2010 Elsevier Inc All rights reserved

引用

页码：75 / 82

页数：8

共 33 条

[1] A combined approach to improving large-scale production of tobacco etch virus protease [J].