An improved strategy for high-level production of TEV protease in Escherichia coli and its purification and characterization.
Because of its stringent sequence specificity, tobacco etch virus (TEV) protease emerges as a useful reagent with wide application in the cleavage of recombinant fusion proteins. However, the solubility of TEV protease expressed in Escherichia coli is extremely low. In the present study, we introduced a more efficient system to improve and facilitate the soluble production of TEV protease in E. coli. Optimal expression of soluble His6-TEV was achieved by examining the contribution of chaperone co-expression and lower temperature fermentation. When further purified by Ni(2+) affinity chromatography, 65mg of His6-TEV was isolated with purity over 95% from 1L of culture. The enzyme activity of His6-TEV was generally characterized by using GST-EGFP and His6-L-TNF fusion protein as substrates, which contained a TEV cleavage site between two moieties.
Fang
Lei
L
The State Key Laboratory of Pharmaceutical Biotechnology and Department of Biochemistry, College of Life Science, Nanjing University, Nanjing 210093, PR China.
Jia
Kun-Zhi
KZ
Tang
Ya-Lan
YL
Ma
Ding-Yuan
DY
Yu
Mei
M
Hua
Zi-Chun
ZC
D018835:Chaperonin 10 / Q000096:biosynthesis; D018834:Chaperonin 60 / Q000096:biosynthesis; D002846:Chromatography, Affinity; D003001:Cloning, Molecular / Q000379:methods*; D010450:Endopeptidases / Q000096:biosynthesis* / Q000302:isolation & purification* / Q000378:metabolism; D004790:Enzyme Induction; D004926:Escherichia coli / Q000201:enzymology; D029968:Escherichia coli Proteins / Q000096:biosynthesis; D006639:Histidine / Q000737:chemistry; D007544:Isopropyl Thiogalactoside / Q000494:pharmacology; D018832:Molecular Chaperones / Q000494:pharmacology; D009842:Oligopeptides / Q000737:chemistry; D019696:Peptidylprolyl Isomerase / Q000096:biosynthesis; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D012995:Solubility; D013696:Temperature
D018835:Chaperonin 10; D018834:Chaperonin 60; D029968:Escherichia coli Proteins; C471213:His-His-His-His-His-His; D018832:Molecular Chaperones; D009842:Oligopeptides; D011993:Recombinant Fusion Proteins; D007544:Isopropyl Thiogalactoside; D006639:Histidine; D010450:Endopeptidases; C099488:TEV protease; C403324:trigger factor, E coli; D019696:Peptidylprolyl Isomerase