A method to obtain high-expression levels of recombinant horseradish peroxidase isozyme C (HRP C) in Spodopterafrugiperda 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. (C) 2004 Elsevier Ltd. All rights reserved.