MIRANDA Maria Victoria
congresos y reuniones científicas
A novel method for expression and purification of recombinant peroxidase in Spodoptera frugiperda larvae.
Barcelona, España
Congreso; 14 th European Congress on Biotechnology; 2009
Horseradish peroxidase (HRP) plays an important role in many biotechnological 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 frugiperdalarvae. The recombinant baculovirus polyhedrin-minus was assembled by using BaculoGoldBrigth that is capable of expressing GFP and the vector pAcGP67 that encodes a sequence for the glycoprotein 67 leader peptide at the 5´end which targets the recombinant protein. This construct allow to monitoring the infection visually under UV illumination. Synthetic HRPC gene was generously provided by Dr. PE Ortiz de Montellano of the University of California. Early fifth-instar S. frugiperda larvae were infected with 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 ly was 100% in all experiments. When an arbitrary threshold was set up at 300 U/ml, 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 1.4 x 107 ufp/ml. Larval extracts at day 7 post-infection expressed 319 ± 56 mg HRPC/Kg larvae while at day 6 post-infection the expression was only 137 ± 17 mg HRPC/Kg larvae. The recombinant HRPC was catalytically active and had a molecular mass comparable to that of the plant enzyme (44 kDa) as judged by SDS-PAGE and Westen Blot, on the basis of this result, it can be assumed that the glycosylation degree should be similar to that of the native HRP (21.8%).  HRP was purified by ion exchange chromatography directly from the larvae extract at pH 7.0 with a yield of 80% and a purity of 70%. The process herein described is cheap and suitable for scaling up.