High level expression of horseradish peroxidase isozyme C in insect larvae.

LOUSTAU, M.; LEVIN, G.; MAGRI, M.L.; TABOGA, O.; MIRANDA, M.V.; CASCONE, O.

Pinamar, Argentina

Congreso; XLI Reunión Anual; 2005

Peroxidase from Armoracia rusticana roots (HRP) is mainly used in medical diagnosis kits. All HRP for local industries must be imported.  As HRP is highly glycosylated, its expression in the active form in prokaryotes is not possible.  The baculovirus-insect cell system is an interesting alternative for its expression.  So far, we have expressed 20 mg/l in  Spodoptera frugiperda cell line cultures.   The enzyme was genetically modified to increase its isoelectric point through a 6xArg fusion tail addition, thus allowing  an easier purification  by  cation-exchange chromatography. Our goal in this work was the design of other alternative expression system in insect larvae (Rachiplusia nu). The recombinant baculovirus contained the HRP6xArg gen under the polyhedrin promoter control and the GP67 secretion signal of Autographa californica. After amplification, a viral clon was used to infect larvaes by subcutaneous inoculation.  At day-3 post-infection hemolymph was collected and extracts of total larvae were prepared. HRP concentration was 105 and 24mg/l respectively.  To keep samples free of melanization, 0.05 M sodium phophate buffer, pH 6.0, EDTA 5 mM, KCl 0.15 M and glutation cristals was selected after assaying different buffers. Native PAGE and IEF with specific staining showed a band corresponding to HRP and pI 9.5 in both samples.