MIRANDA Maria Victoria
Expression and purification of horseradish peroxidase in insect larvae
LOUSTAU, M; ROMERO, L; LEVIN, G; MAGRI, M; LÓPEZ, M.,; TABOGA, O.,; CASCONE, O; MIRANDA, M.V.
Año: 2008 vol. 43 p. 103 - 103
Insect cells infected with recombinant baculovirus represent an interesting system to achieve high level expression of commercially attractive proteins like immunogens and diagnostic reagents. The aim of this work is to study different downstream processing strategies for the recovery of a recombinant biocatalyst (horseradish peroxidasa, HRPr) directly from the Sf9 cell culture. We constructed a baculovirus vector by cotransfection of transfer vector and viral DNA BaculoGoldTM brigth in Sf9 cell line. The cDNA of HRP supports two tags (6xHis and 6xArg) and was cloned under the polyhedrin promoter and the signal peptide sequence GP67. A viral stock of 9.2 x107 pfu.ml-1 was used to infect the suspension cell cultures. HRPr was secreted into the culture medium where it acumulated to 11.6 mg.l-1 at 5 days postinfection with MOI 2 and the supplementation of the medium with 7.2 ìM hemin. HRPr was purified by nickel affinity chromatography (IMAC) and by ion exchange chromatography (IEC) directly from the clarified supernatant cultures. In the first case, HRPr yield was 96% with a purification factor of 60.4 and in IEC at pH 8.5 the yield was 89.9% with a purification factor of 24. High yields of HRPr were achieved by both strategies but IEC has lower cost and therefore is more suitable for scaling up the process.