High level expression of horseradish peroxidase isozyme C in insect larvae

LOUSTAU, M. N.; GUSTAVO LEVIN,; MAGRI, MARÍA L.; MIRANDA, MARÍA V; OSVALDO CASCONE

Pinamar

Congreso; XLI Reunión Anual de la Sociedad Argentina de Investigación en Bioquímica y Biología Molecular; 2005

SAIB

  HIGH LEVEL EXPRESSION OF HORSERADISH PEROXIDASE ISOZYME C IN INSECT LARVAE   Loustau, María N.; Levin, Gustavo; Magri, María L.;  Miranda, María V. and Cascone, Osvaldo Cátedra de Microbiología Industrial y Biotecnología. Facultad de Farmacia y Bioquímica. UBA. Junín 956 (1113) Buenos Aires. E-mail: mnloustau@ffyb.uba.ar   Peroxidase from Amoracia rusticana roots (HRP) is an enzyme mainly used in medical diagnosis kits. All HRP for local industries must be imported. HRP is highly glycosylated, therefore its production as a recombinant protein 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 an 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 clone was used to infect larvae by subcutaneous inoculation. At day-3 post-infection hemolimph was collected and extracts of total larvae were prepared. HRP concentration was 1.6 and 0.8 mg/g respectively. To keep samples free of melanization, 0.05 M sodium phosphate buffer, pH 6.0, 5 mM EDTA, 0.15 M KCl and glutation crystals was selected. Native PAGE and IEF with specific staining showed a band corresponding to the molecular weight of HRP and pI 9.5 in both samples.