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Targovnik A.M.,Catedra de Microbiologia Industrial y Biotecnologia | Cascone O.,Catedra de Microbiologia Industrial y Biotecnologia | Miranda M.V.,Catedra de Microbiologia Industrial y Biotecnologia
Separation and Purification Technology | Year: 2012

Aqueous two-phase systems (ATPSs) have not yet been applied to purify proteins expressed in insect larvae infected by recombinant baculovirus. This work describes the behavior of typical contaminants in the baculovirus-insect larvae expression system such as larval proteins and baculovirus particles in PEG/phosphate ATPSs, in addition to the extraction and purification of the target protein (horseradish peroxidase isozyme C, HRPC). After assessing the influence of PEG molecular weight, system pH and added salt on the partition constants of HRPC and total protein of a clarified larvae extract, two ATPSs were selected for the first extraction step: PEG 1500/phosphate, pH 7.0 with 4.0% NaCl (System 1) and PEG/phosphate, pH 5.0 in the absence of NaCl (System 2). Both systems were found to be appropriate since a clarified enzyme-enriched top phase was obtained with a yield of 99% and 90% respectively. The direct partition of larvae homogenized with the components of Systems 1 and 2, yielded a HRPC recovery in top phase of 71.4% and 81.1% respectively, whereas total protein recovery was 5.2% and 3.3% respectively. In both systems, the top phase was clear and particulate material remained in the interphase and the bottom phase. The bulk of immunogenic proteins of the larvae concentrated in the bottom phase of both systems. The PCR assay revealed the presence of viral DNA in both phases. It was possible to extract the HRPC back from the PEG-rich phase by adding a fresh magnesium sulfate solution to form a new ATPS, achieving a recovery in the bottom phase of 50% and 98% in Systems 1 and 2 respectively, whereas the recovery of total protein was 69% and 24% respectively. The HRPC global recovery of the two-step processes was 35.4% and 79.6% for Systems 1 and 2, with purification factors of 14.5 and 114.2 respectively. The final product was free of viral particles. © 2012 Elsevier B.V. All rights reserved. Source


Romero L.,Catedra de Microbiologia Industrial y Biotecnologia | Targovnik A.,Catedra de Microbiologia Industrial y Biotecnologia | Wolman F.,Catedra de Microbiologia Industrial y Biotecnologia | Fogar M.,Instituto Nacional de Tecnologia Agropecuaria | And 3 more authors.
New Biotechnology | Year: 2010

Horseradish peroxidase isozyme C (HRPC) is an important commercial biocatalyst. In this study, a screening of different lepidopteran species frequently found in Argentina to produce this protein was carried out. Two recombinant viruses were constructed: AcMNPV HRPC polyhedrin-minus (occ-), an intrahemocelical infective virus; and AcMNPV HRPC polyhedrin-plus (occ+), to achieve an oral infective baculovirus. Each lepidopteran species was infected either with AcMNPV HRPC occ- or AcMNPV HRPC occ+ and the harvesting days post-infection (dpi) were optimized. All species were susceptible to AcMNPV HRPC occ- infection, giving Spodoptera frugiperda the best yield: 41 μg per larva. Rachiplusia nu was highly susceptible to oral infection, reaching 22 μg per larva at 4. dpi. HRPC was purified by IMAC from S. frugiperda extracts with a yield of 86% and a purification factor of 29. © 2010 Elsevier B.V. Source


Targovnik A.M.,Catedra de Microbiologia Industrial y Biotecnologia | Romero L.V.,Catedra de Microbiologia Industrial y Biotecnologia | Wolman F.J.,Catedra de Microbiologia Industrial y Biotecnologia | Cascone O.,Catedra de Microbiologia Industrial y Biotecnologia | Miranda M.V.,Catedra de Microbiologia Industrial y Biotecnologia
Process Biochemistry | Year: 2010

Horseradish peroxidase is used in many biotechnological fields including diagnostics, biocatalysts and biosensors. Horseradish peroxidase isozyme C (HRPC) was extracellularly expressed in Spodoptera frugiperda Sf9 cell culture and in intact larvae. At day 6 post-infection, the concentration of active HRPC in suspension cultures was 3.0 ± 0.1 μg per 1 × 106 cells or 3.0 ± 0.1 mg l-1 with a multiplicity of infection of 1 in the presence of 7.2 μM hemin. Similar yields were obtained in monolayer cultures. In larvae, the HRPC expression level was 137 ± 17 mg HRPC kg-1 larvae at day 6 post-infection with a single larvae thus producing approximately 41 μg HRPC. The whole larval extract was separated by ion exchange chromatography and HRPC was purified in a single step with a yield of 75% and a purification factor of 117. The molecular weight of recombinant HRPC was 44,016 Da, and its glycosylation pattern agreed with that expected for invertebrates. The Km and Vmax were 12.1 ± 1.7 mM and 2673 ± 113 U mg-1, respectively, similar to those of HRP purified from Armoracia rusticana roots. The method described in this study, based on overexpression of HRPC in S. frugiperda larvae, is a simple and inexpensive way to obtain high levels of active enzyme for research and other biotechnological applications. © 2010 Elsevier Ltd. All rights reserved. Source


Romero L.V.,Catedra de Microbiologia Industrial y Biotecnologia | Targovnik A.M.,Catedra de Microbiologia Industrial y Biotecnologia | Wolman F.J.,Catedra de Microbiologia Industrial y Biotecnologia | Cascone O.,Catedra de Microbiologia Industrial y Biotecnologia | Miranda M.V.,Catedra de Microbiologia Industrial y Biotecnologia
Biotechnology Letters | Year: 2011

A process based on orally-infected Rachiplusia nu larvae as biological factories for expression and one-step purification of horseradish peroxidase isozyme C (HRP-C) is described. The process allows obtaining high levels of pure HRP-C by membrane chromatography purification. The introduction of the partial polyhedrin homology sequence element in the target gene increased HRP-C expression level by 2. 8-fold whereas it increased 1. 8-fold when the larvae were reared at 27°C instead of at 24°C, summing up a 4. 6-fold overall increase in the expression level. Additionally, HRP-C purification by membrane chromatography at a high flow rate greatly increase D the productivity without affecting the resolution. The V max and K m values of the recombinant HRP-C were similar to those of the HRP from Armoracia rusticana roots. © 2011 Springer Science+Business Media B.V. Source

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