Purification challenge in baculovirus-insect larvae expression system

LOUSTAU, MARÍA DE LAS NIEVES; LEVIN, GUSTAVO JAVIER; ROMERO, LUCÍA VIRGINIA; NAVARRO DEL CANIZO, AGUSTIN A.; MIRANDA, MARÍA VICTORIA; CASCONE, OSVALDO

Rosario

Congreso; XLII Reunión Nacional de la Sociedad Argentina de Investigación en Bioquímica y Biología Molecular; 2006

SAIB

  Horseradish peroxidase (HRP) is widely used in immunology diagnosis kits. As its source is the Amoracia rusticana root (horseradish), a poor developed culture in Argentina, this enzyme must be imported. Our goal was to express HRP as a recombinant enzyme and purify it up to the analytical purity level. Due to its structural complexity (highly glycosylated heme protein, stabilized by four disulfide bonds), its expression in active form in prokaryote systems is not possible. A low-cost alternative is the baculovirus-cell insect system. Previously, recombinant HRP was obtained in insect cell cultures and, for the scale-up of the process, the same virus was used to infect Rachiplusia nu larvae. Though high peroxidase levels were reached (115.9 ± 8.2 mg/kg larvae), the contamination by larva proteins was the main problem to solve, in particular catalase (CAT), enzyme that competes with HRP for hydrogen peroxide.  Since the recombinant enzyme has a 6xHis and a 6xArg tags, immobilized metal ion affinity chromatography (IMAC) and ion exchange chromatography (IEC) were assayed. As HRP accumulates in haemolymph, but haemolymph is difficult to withdraw quantitatively, total larvae extract was chosen as the starting material. SDS-PAGE shows that both methods are able to separate HRP from most of larvae contaminating proteins and CAT, with enzyme yields over 97 %, being IEC more economic.