Genetic engineering applied to recombinant peroxidase purification by ion exchange chromatography

LEVIN, G. J.; TABOGA, O.; NAVARRO DEL CAÑIZO, A.; MENDIVE, F. M.; TARGOVNIK, H. M.; CASCONE, O.; MIRANDA, M. V.

San Miguel de Tucumán. Argentina

Simposio; Simposio Internacional de Biotecnología y II Simposio Argentino-Italiano de Bacterias Lácticas; 2004

ABSTRACT A genetically engineered HRP C isoenzyme C gene was constructed through the addition of charged polypeptide fusion tail by PCR strategy. HRP C-6x arg cDNA was expressed in insect-baculovirus system under polyhedrin promotor and in E. coliE. coli BL21(DE3) codon plus. Recombinant peroxidase incremented its isoelectric point that was major than 9.5 as judged by isoelectric focusing. cDNA was cloned into both vectors: pRSET A for prokaryota expression and in pAcGP67B, a transfer vector to recombinant baculovirus construction. After its purification from culture supernatant in Sf9 cell culture a yield of 98.5% and a purification factor of 130 was obtained. In the case of prokariota expression, the enzyme appears as inactive protein forming inclusion bodies as judge by Western. In this case the yield was 96% with high degree of purity.