Facilitated downstream processing of recombinant polyarg-peroxidase from insect cell-baculovirus system.

LEVIN, G.; PEREDA, A.; TABOGA, O.; CASCONE, O.; MIRANDA, M.V.

Río de Janeiro, Brasil

Congreso; EMPROMER; 2005

  Downstream processing of biotechnological products is facilitated utilizing novel operations for the direct capture of the target entity. It was recently shown that fluidized strongly acidic cation exchanger beads performed well during protein recovery from complex biomass suspensions. Enhancement of this technology will result from increased selectivity –at the molecular level- of the adsorption process. The aim of this work was to design a model fusion product, horseradish peroxidase isozyme c (HRPc), in order to promote selective binding of the same to adsorbent particles. Different genetically engineered HRPc enzymes was constructed through the addition of six or eight arginine residues at C terminal.  The products were expressed intra and extracellular in different cell lines (Sf9, Sf21 and High Five) and purified directly from expression medium or cell lysates. The isoelectric point of enzyme was elevated up to 9.0. After ion exchange purification a recombinant HRPc yield of 98.5 % 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 utilizing novel operations for the direct capture of the target entity. It was recently shown that fluidized strongly acidic cation exchanger beads performed well during protein recovery from complex biomass suspensions. Enhancement of this technology will result from increased selectivity –at the molecular level- of the adsorption process. The aim of this work was to design a model fusion product, horseradish peroxidase isozyme c (HRPc), in order to promote selective binding of the same to adsorbent particles. Different genetically engineered HRPc enzymes was constructed through the addition of six or eight arginine residues at C terminal.  The products were expressed intra and extracellular in different cell lines (Sf9, Sf21 and High Five) and purified directly from expression medium or cell lysates. The isoelectric point of enzyme was elevated up to 9.0. After ion exchange purification a recombinant HRPc yield of 98.5 % 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.