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

D. M. Hushpulian¹, P. A. Savitski², A. M. Rojkova¹, T. A. Chubar¹, V. A. Fechina², I. Yu. Sakharov¹, L. M. Lagrimini³, V. I. Tishkov^1*, and I. G. Gazaryan¹

¹Department of Chemical Enzymology, Faculty of Chemistry, Lomonosov Moscow State University, Moscow 119992, Russia; fax: (7-095) 939-2742; E-mail: vit@enz.chem.msu.ru; igazaryan@hotmail.com

²Bach Institute of Biochemistry, Russian Academy of Sciences, Leninsky pr. 33, Moscow 119071, Russia; fax: (7-095) 954-2804; E-mail: zherdev@inbi.ras.ru

³Syngenta Biotechnology, 3054 Cornwallis Rd., Research Triangle Park, NC 27709-2257, USA; fax: (919) 541-8585; E-mail: mark.lagrimini@syngenta.com

^* To whom correspondence should be addressed.

Received June 24, 2003; Revision received July 28, 2003

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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 ~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.

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KEY WORDS: recombinant tobacco peroxidase, expression, refolding, purification, calcium effect

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