title: High-yield reactivation of anionic tobacco peroxidase overexpressed in Escherichia coli.
abstract: 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 μg/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 μM 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).
authors.affiliation: A.N. Bach Institute of Biochemistry, RAS, 119071 Moscow, Russia; Innovations and High Technologies MSU Ltd, 109559 Moscow, Russia. Electronic address: galina.s.zakharova@gmail.com.
authors.forename: G S
authors.initials: GS
authors.lastname: Zakharova
authors.affiliation: Innovations and High Technologies MSU Ltd, 109559 Moscow, Russia; M.V. Lomonosov Moscow State University, Chemistry Faculty, Department of Chemical Enzymology, 119899 Moscow, Russia.
authors.forename: A A
authors.initials: AA
authors.lastname: Poloznikov
authors.affiliation: M.V. Lomonosov Moscow State University, Chemistry Faculty, Department of Chemical Enzymology, 119899 Moscow, Russia.
authors.forename: T A
authors.initials: TA
authors.lastname: Chubar
authors.affiliation: M.V. Lomonosov Moscow State University, Chemistry Faculty, Department of Chemical Enzymology, 119899 Moscow, Russia.
authors.forename: I G
authors.initials: IG
authors.lastname: Gazaryan
authors.affiliation: A.N. Bach Institute of Biochemistry, RAS, 119071 Moscow, Russia; Innovations and High Technologies MSU Ltd, 109559 Moscow, Russia; M.V. Lomonosov Moscow State University, Chemistry Faculty, Department of Chemical Enzymology, 119899 Moscow, Russia.
authors.forename: V I
authors.initials: VI
authors.lastname: Tishkov
mesh_terms: D004926:Escherichia coli / Q000235:genetics*; D006427:Hemin; D006863:Hydrogen-Ion Concentration; D002479:Inclusion Bodies; D010544:Peroxidases / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D058849:Protein Refolding; D011994:Recombinant Proteins / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D014508:Urea
chemical_list: D011994:Recombinant Proteins; D006427:Hemin; D014508:Urea; D010544:Peroxidases; C085547:tobacco anionic peroxidase
keywords: Anionic tobacco peroxidase; Escherichia coli; Inclusion bodies; Protein refolding; Recombinant protein expression