Ingeniería genética aplicada a la purificación de peroxidasa recombinante por intercambio iónico

GUSTAVO LEVIN,; NAVARRO DEL CANIZO, AGUSTIN A.; TARGOVNIK, HÉCTOR M; OSVALDO CASCONE; MIRANDA, MARÍA V

San Miguel de 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 by the PCR strategy. HRP C-6xArg 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 purification from Sf9 cell culture supernatant, a HRP C yield of 98.5% with a purification factor of 130 was obtained. 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.