INVESTIGADORES
MIRANDA Maria Victoria
congresos y reuniones científicas
Título:
Purification challenge in baculovirus-insect larvae expression system
Autor/es:
LOUSTAU, M.; LEVIN, G.; ROMERO, L.; NAVARRO, A.; MIRANDA, M.V.; CASCONE, O.
Lugar:
Rosario, Argentina
Reunión:
Congreso; XLII Reunión Anual SAIB; 2006
Resumen:
  Horseradish peroxidase (HRP) is widely used in laboratory assays, particularly in immunology diagnosis kits. As its traditional source is the Amoracia rusticana root (horseradish), this enzyme is not produced in Argentina. Our goal is to express HRP as a recombinant enzyme. Due to its structural complexity (high glycosylated heme enzyme, stabilized by four disulfide bonds), its expression in prokaryote systems is not possible. A low cost alternative is baculovirus-cell insect system. In a primary phase the recombinant protein was obtained in cell cultures, but with the aim of scaling this process, the same virus was used to infect Rachiplusia nu larvae. Though high production levels were reached (115.9 ± 8.2 mg/kg larvae), the contamination with larvae proteins was a main problem to solve, in particular. Catalase (CAT), as this enzyme competes with HRP for hydrogen peroxide. Since the recombinant enzyme has a 6xHis and 6xArg tags, immobilized metal affinity chromatography (IMAC) and ionic exchange chromatography were assayed. As HRP accumulated in haemolymph, but haemolymph is difficult to withdraw quantitatively, total larvae extract was chosen as started material. SDS-PAGE  shows that both methods are able to separate HRP from most of contaminaiting proteins and CAT, with enzyme yield over 97% bein IEC more economic.