High-yield reactivation of anionic tobacco peroxidase overexpressed in Escherichia coli

Zakharova, G.S.; Poloznikov, A.A.; Chubar, T.A.; Gazaryan, I.G.; Tishkov, V.I.

Авторы: Zakharova G.S., Poloznikov A.A., Chubar T.A., Gazaryan I.G., Tishkov V.I.
Журнал: Protein Expression and Purification
Том: 113
Номер: 1
Год издания: 2015
Издательство: Academic Press
Местоположение издательства: United States
Первая страница: 85
Последняя страница: 93
DOI: 10.1016/j.pep.2015.05.007
Аннотация: Anionic tobacco peroxidase (TOP) is extremely active in chemiluminescence reaction of luminol oxidation without addition of enhancers and more stable than horseradish peroxidase under antibody conjugation conditions. In addition, recombinant TOP (rTOP) produced in Escherichia coli is known to be a perfect direct electron transfer catalyst on electrodes of various origin. These features make the task of development of a high-yield reactivation protocol for rTOP practically important. Previous attempts to reactivate the enzyme from E. coli inclusion bodies were successful, but the reported reactivation yield was only 14%. In this work, we thoroughly screened the refolding conditions for dilution protocol and compared it with gel-filtration chromatography. The impressive reactivation yield in the dilution protocol (85%) was achieved for 8 lg/mL solubilized rTOP protein and the refolding medium containing 0.3 mM oxidized glutathione, 0.05 mM dithiothreitol, 5 mM CaCl2, 5% glycerol in 50 mM Tris–HCl buffer, pH 9.6, with 1 lM hemin added at the 24th hour of incubation. A practically important discovery was a 30–40% increase in the reactivation yield upon delayed addition of hemin. The reactivation yield achieved is one of the highest reported in the literature on protein refolding by dilution. The final yield of purified active non-glycosylated rTOP was ca. 60 mg per L of E. coli culture, close to the yield reported before for tomato and tobacco plants overexpressing glycosylated TOP (60 mg/kg biomass) and much higher than for the previously reported refolding protocol (2.6 mg per L of E. coli culture).
Добавил в систему: Тишков Владимир Иванович

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High-yield reactivation of anionic tobacco peroxidase overexpressed in Escherichia coliстатья

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