INVESTIGADORES
LEVIN Gustavo Javier
congresos y reuniones científicas
Título:
Facilitated downstream processing of recombinant polyarg-peroxidase from insect cell-baculovirus system
Autor/es:
LEVIN, G. J.; PEREDA, A.; TABOGA, O.; CASCONE, O.; MIRANDA, M. V.
Lugar:
Costa Verde, Rio de Janeiro. Brasil
Reunión:
Congreso; 2nd Mercosur Congress on Chemical Engineering. 4th Mercosur Congress on Process Systems Engineering. ENPROMER.; 2005
Resumen:
Abstract. Downstream processing of biotechnological products is facilitated by utilising strategies such as
isoelectric point rise by a polyarginine tag fusion for capturing the target entity directly from the culture
medium. It was shown that fluidized strongly acidic cation exchanger beads performed well during protein
recovery from complex biomass suspensions. Application of cation-exchange chromatography at high pH will
result in an increased selectivity for biomolecules of high isoelectric point. The aim of this work was to
design a model fusion product, horseradish peroxidase isozyme c (HRPc), in order to increase its isoelectric
point, thus promoting selective binding to fluidised cation exchangers at high pH. A genetically engineered
HRPc enzyme was constructed through the addition of six arginine residues at C terminus. The products
were expressed extracellularly in different cell lines (Sf9, Sf21) and purified directly from the expression
medium. The isoelectric point of enzyme was raised to over 9.0. After ion exchange purification at pH 8.5, a
recombinant HRPc yield over 98 % with a purification factor of 130 was obtained. This creates the
opportunity for facilitated direct recovery of recombinant products employing fluidised cation exchangers
under selective process conditions.. Downstream processing of biotechnological products is facilitated by utilising strategies such as
isoelectric point rise by a polyarginine tag fusion for capturing the target entity directly from the culture
medium. It was shown that fluidized strongly acidic cation exchanger beads performed well during protein
recovery from complex biomass suspensions. Application of cation-exchange chromatography at high pH will
result in an increased selectivity for biomolecules of high isoelectric point. The aim of this work was to
design a model fusion product, horseradish peroxidase isozyme c (HRPc), in order to increase its isoelectric
point, thus promoting selective binding to fluidised cation exchangers at high pH. A genetically engineered
HRPc enzyme was constructed through the addition of six arginine residues at C terminus. The products
were expressed extracellularly in different cell lines (Sf9, Sf21) and purified directly from the expression
medium. The isoelectric point of enzyme was raised to over 9.0. After ion exchange purification at pH 8.5, a
recombinant HRPc yield over 98 % with a purification factor of 130 was obtained. This creates the
opportunity for facilitated direct recovery of recombinant products employing fluidised cation exchangers
under selective process conditions.