A new tagged-TEV protease: construction, optimisation of production, purification and test activity.
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 coli 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°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. coli KRX strain under an induction temperature of 25°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.
Miladi
Baligh
B
Laboratory of Molecular Biology, Ecole de Biologie Industrielle, 32 Bd du Port, Cergy 95094, France.
Bouallagui
Hassib
H
Dridi
Cyrine
C
El Marjou
Ahmed
A
Boeuf
Guilhem
G
Di Martino
Patrick
P
Dufour
Florence
F
Elm'Selmi
Abdellatif
A
D001483:Base Sequence; D003001:Cloning, Molecular; D010450:Endopeptidases / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D004926:Escherichia coli / Q000235:genetics*; D015870:Gene Expression; D005822:Genetic Vectors / Q000235:genetics; D006639:Histidine / Q000235:genetics*; D008969:Molecular Sequence Data; D009842:Oligopeptides / Q000235:genetics*; D010942:Plant Viruses / Q000201:enzymology* / Q000235:genetics; D010957:Plasmids / Q000235:genetics; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D014026:Nicotiana / Q000821:virology; D014764:Viral Proteins / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism
C471213:His-His-His-His-His-His; D009842:Oligopeptides; D011993:Recombinant Fusion Proteins; D014764:Viral Proteins; D006639:Histidine; D010450:Endopeptidases; C099488:TEV protease