Expression and Refolding of Tobacco Anionic Peroxidase from E. coli Inclusion Bodies

Abstract

Coding DNA of the tobacco anionic peroxidase gene was cloned in pET40b vector. The problem of 11 arginine codons, rare in procaryotes, in the tobacco peroxidase gene was solved using E. coli BL21(DE3) Codon Plus strain. The expression level of the tobacco apo-peroxidase in the above strain was approximately 40% of the total E. coli protein. The tobacco peroxidase refolding was optimized based on the earlier developed protocol for horseradish peroxidase. The reactivation yield of recombinant tobacco enzyme was about 7% with the specific activity of 1100-1200 U/mg towards 2,2;-azino-bis(3-ethylbenzothiazoline-6-sulfonate) (ABTS). It was shown that the reaction of ABTS oxidation by hydrogen peroxide catalyzed by recombinant tobacco peroxidase proceeds via the ping-pong kinetic mechanism as for the native enzyme. In the presence of calcium ions, the recombinant peroxidase exhibits a 2.5-fold decrease in the second order rate constant for hydrogen peroxide and 1.5-fold decrease for ABTS. Thus, calcium ions have an inhibitory effect on the recombinant enzyme like that observed earlier for the native tobacco peroxidase. The data demonstrate that the oligosaccharide part of the enzyme has no effect on the kinetic properties and calcium inhibition of tobacco peroxidase.

Tobacco Peroxidase as a New Reagent for Amperometric Biosensors

“…(3) A comparison between data for native and recombinant HRPs indicates that the deglycosylation of the enzyme significantly improved all of the characteristics of an enzyme electrode under conditions of both direct electron transfer and mediated electron transfer. Thus, the preparation of unglycosylated recombinant TOP in our laboratory [2] is of undoubted interest for its use in biosensors.…”

“…Thus, the tests of TOP in various amperometric biosensors demonstrated that the new enzyme is superior to HRP in a number of parameters; this enzyme is of great interest to the development of new analytical methods. A further improvement in parameters and the design of new biosensors based on TOP can be implemented usinga recently prepared unglycosylated recombinant form of the enzyme [2]. In addition to the designs of biosensors considered in this survey, genetically engineered TOP can be used in amperometric flow immunosensors based on a socalled P-chip (peroxidase chip) principle [23] and enzyme tunneling [24].…”

“…Recently, with the use of TOP gene expression in the cells of E. coli, a genetically engineered unglycosylated enzyme was obtained in amounts sufficient for crystallization, other resource-demanding physicochemical investigation techniques, and testing for analytical purposes [2]. Thus, our laboratory is the only world's laboratory capable of producing genetically engineered (recombinant) HRP and recombinant TOP in gram amounts.…”

Molecular Cloning and Partial Characterization of a Peroxidase Gene Expressed in the Roots ofPortulaca oleraceacv., One Potentially Useful in the Remediation of Phenolic Pollutants

“…23,24) Using the BY2 system, we were able to produce active HRP C1a containing N-glycans. 14) In addition, to simplify purification, PRX protein can be secreted into the culture medium of BY2 cells.…”

Horseradish and soybean peroxidases: comparable tools for alternative niches?

“…Henriksen and 110 co-workers [15] also developed a recombinant SBP for crystallisation studies based 111 on previous cDNA work, in which they successfully refolded SBP from inclusion 112 bodies using a cocktail that included both oxidized and reduced glutathione [27]. 113 There have been several other recent examples of recombinantly expressed plant 114 peroxidases including Hushpulian and co-workers' [28] work on tobacco anionic 115 peroxidase. 116…”