CONICET | Buscador de Institutos y Recursos Humanos

Horseradish peroxidase (HRP) plays an important role in manybiotechnological fields, including diagnostics, biocatalysis and biosensors. The aim of this work was to optimize a process for expression and purification of HRP isozyme C (HRP C) in Spodoptera frugiperda larvae. The recombinant baculovirus polvhedrin-minus was assembled by using BaculoGoldBrigth that is capable of expressing GFP and the vector pAcGP6T that encodes for the glycoprotein 67 leader peptide which targets the recombinant protein. This construct allows monitoring the infection visually under UV illumination. Synthetic HRPC gene was generously provided by Dr. P.E. Ortiz de Montellano of the University of California. Early fifth-instar S. frugiperda larvae were infected with the recombinant virus. A direct correlation between GFP and HRPC expression profile exists, this facilitating the selection of larvae to process from day 3-post infection. The efficiency of intrahaemocoelical infection via was 100% in all experiments. When an arbitrary threshold of 300U/mI was set up, 50% of the larvae exceeded this reference value at day 5, 85% at day 6 and 93% at day 7. The optimum viral titre was l x 107 ufp/ml. Larval extracts at day 7 post-infection expressed 319 ± 56mg HRPC/kg larvae while at day 6 post-infection the expression was only 137±1,7mg HRPC/kg larvae. The recombinant HRPC is catalytically active and its molecular mass is comparable to that of the plant enzyme (44 kDa) as judged by SDS-PAGE and Western blot; on the basis of this result it can be assumed that the glycosylation degree should be similar to that of the nativeHRP (21.87%). HRPC was purified by ion exchange chromatography directly from the larvae extract at pH 7.0 with a yield of 80% and a purity of 7%. The process herein described is cost-effective and suitable for scaling up.

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