GENETIC ENGINEERING APPLIED TO RECOMBINANT PEROXIDASE PURIFICATION BY ION EXCHANGE CHROMATOGRAPHY

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

Tucumán

Congreso; Simposio Internacional de Biotecnología; 2004

A genetically engineered horseradish peroxidase isoenzyme C (HRP C) gene was constructed by the addition of a charged polypeptide fusion tail to the 6xHis-HRP C by the PCR strategy. 6xHis-6xArg-HRP C cDNA was expressed in an insect-baculovirus system under polyhedrin promoter and in E. coli BL21(DE3) codon plus. Recombinant peroxidase isoelectric point was raised to over 9.5 as judged by isoelectric focusing. cDNA was cloned in two vectors: pRSET A for prokaryote expression and pAcGP67B, a transfer vector for recombinant baculovirus construction. After ion exchange purification from a Sf9 cell culture supernatant, a recombinant HRP C yield of 98.5% with a purification factor of 130 was obtained, while IMAC purification yield was only 68 % with a similar purification factor. In the case of prokaryote expression, the enzyme was in inclusion bodies, and could be obtained in a 96% yield with a high purity degree by direct loading of the 8 M urea-dissolved to an ion exchanger. E. coli BL21(DE3) codon plus. Recombinant peroxidase isoelectric point was raised to over 9.5 as judged by isoelectric focusing. cDNA was cloned in two vectors: pRSET A for prokaryote expression and pAcGP67B, a transfer vector for recombinant baculovirus construction. After ion exchange purification from a Sf9 cell culture supernatant, a recombinant HRP C yield of 98.5% with a purification factor of 130 was obtained, while IMAC purification yield was only 68 % with a similar purification factor. In the case of prokaryote expression, the enzyme was in inclusion bodies, and could be obtained in a 96% yield with a high purity degree by direct loading of the 8 M urea-dissolved to an ion exchanger. Sf9 cell culture supernatant, a recombinant HRP C yield of 98.5% with a purification factor of 130 was obtained, while IMAC purification yield was only 68 % with a similar purification factor. In the case of prokaryote expression, the enzyme was in inclusion bodies, and could be obtained in a 96% yield with a high purity degree by direct loading of the 8 M urea-dissolved to an ion exchanger.