GENETIC ENGINEERING APPLIED TO RECOMBINANT PEROXIDASE PURIFICATION BY ION EXCHANGE CHROMATOGRAPHY.

LEVIN, GUSTAVO J.; TABOGA, O; NAVARRO DE CAÑIZO, A. A.; MENDIVE, FERNANDO M.; TARGOVNIK, HECTOR M.; CASCONE, OSVALDO; MIRANDA, MARIA V.

San Miguel de Tucumán, Argentina

Simposio; Simposio Internacional de Biotecnología. II Simposio Argentino-Italiano de Bacterias Lácticas; 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. co/i 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 ITom a Sj9 cell culture supematant, a recombinant HRP C yield of 98.5% with a purifjcation factor of 130 was obtained, while IMAC purification yield was only 68 % wih 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 8M urea­ dissolved to an ion exchanger.