info:eu-repo/semantics/article
High-level expression and purification of recombinant horseradish peroxidase isozyme C in SF-9 insect cell culture
Fecha
2005-02Registro en:
Segura, María De Las M.; Levin, Gustavo Javier; Miranda, María V.; Mendive, Fernando M.; Targovnik, Hector Manuel; et al.; High-level expression and purification of recombinant horseradish peroxidase isozyme C in SF-9 insect cell culture; Elsevier; Process Biochemistry; 40; 2; 2-2005; 795-800
0032-9592
CONICET Digital
CONICET
Autor
Segura, María De Las M.
Levin, Gustavo Javier
Miranda, María V.
Mendive, Fernando M.
Targovnik, Hector Manuel
Cascone, Osvaldo
Resumen
A method to obtain high-expression levels of recombinant horseradish peroxidase isozyme C (HRP C) in Spodoptera frugiperda Sf-9 cell culture and a strategy for its purification are described. HRP C was secreted into the culture medium where it accumulated to 25.6 mg/l. Addition of hemin to the insect cell culture increased the level of active enzyme expression up to 41.3 mg/l. A selective staining procedure using 3,3′-diaminobenzidine allowed visualisation of HRP C in the infected insect cells and provided an alternative staining strategy for titration of recombinant baculovirus carrying the HRP gene. Immobilised metal ion affinity chromatography using a Ni-NTA matrix with elution in the gradient-step mode yielded a 68% HRP C recovery with a RZ of 2.8. When the displacement elution mode was utilised, the yield was essentially the same and the product was electrophoretically pure, having a RZ of 3.2.