FACILITATED DOWNSTREAM PROCESSING OF RECOMBINANT

LEVIN, G; PEREDA, A.; TABOGA, O.; CASCONE O; MIRANDA, MARÍA VICTORIA

Argentina

Congreso; ENPROMER 2005; 2005

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. 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.