High-level expression and purification of recombinant horseradish peroxidase isozyme C in SF-9 insect cell culture

Segura, María de las Mercedes; Levin, Gustavo Javier; Miranda, Magdalena; Mendive, Fernando; Targovnik, Héctor M.; Cascone, Osvaldo

doi:10.1016/j.procbio.2004.02.009

Cited by 21 publications

(29 citation statements)

References 11 publications

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“…HRP-C is a single peptide chain with a molar mass of about 44 kDa and an isoelectric point in the range of 8.5-9.0 (32). The presence of a distinct single band for our purified HRP specimen ( Figure 2) confirms that our preparation contains a monomeric protein.…”

Section: Discussionsupporting

confidence: 57%

Purification of Peroxidase from Horseradish (Armoracia rusticana) Roots

Lavery

MacInnis

MacDonald

et al. 2010

J. Agric. Food Chem.

106

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Peroxidase (EC 1.11.1.7) from horseradish ( Armoracia rusticana ) roots was purified using a simple, rapid, three-step procedure: ultrasonication, ammonium sulfate salt precipitation, and hydrophobic interaction chromatography on phenyl Sepharose CL-4B. The preparation gave an overall yield of 71%, 291-fold purification, and a high specific activity of 772 U mg(-1) protein. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed that the purified enzyme was homogeneous and had a molecular weight of approximately 40 kDa. The isolated enzyme had an isoelectric point of 8.8 and a Reinheitszahl value of 3.39 and was stable when stored in the presence of glycerol at -20 degrees C, with >95% retention of original enzyme activity for at least 6 months. Maximal activity of purified horseradish peroxidase (HRP) was obtained under different optimized conditions: substrate (guaiacol and H(2)O(2)) concentrations (0.5 and 0.3 mM, respectively), type of buffer (50 mM phosphate buffer), pH (7.0), time (1.0 min), and temperature of incubation (30 degrees C). In addition, the effect of HRP and H(2)O(2) in a neutral-buffered aqueous solution for the oxidation of phenol and 2-chlorophenol substrates was also studied. Different conditions including concentrations of phenol/2-chlorophenol, H(2)O(2), and enzyme, time, pH, and temperature were standardized for the maximal activity of HRP with these substrates; under these optimal conditions 89.6 and 91.4% oxidations of phenol and 2-chlorophenol were obtained, respectively. The data generated from this work could have direct implications in studies on the commercial production of this biotechnologically important enzyme and its stability in different media.

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Section: Discussionsupporting

confidence: 57%

Purification of Peroxidase from Horseradish (Armoracia rusticana) Roots

Lavery

MacInnis

MacDonald

et al. 2010

J. Agric. Food Chem.

106

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“…2a, illustrating that there is only one substance. Its molecular weight is about 44 kDa, indicating that this peak is the pure HRP peak (Segura et al 2005). The enzymatic activity index (Rz value = A 403 /A 280 ) is about 3.1.…”

Section: Resultsmentioning

confidence: 94%

The formation of a new horseradish peroxidase binding rare earth

Jiang

Wang

et al. 2009

Environ Chem Lett

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The charge distribution, the isolation, purification, and characterization of horseradish peroxidase (HRP) were investigated. A new HRP protein binding La (La-HRP) was found for the first time in vivo. The molecular weight of the La-HRP protein is about 43,833 Da. The activity index (Rz) of the La-HRP protein (Rz = 2.4) is lower than that of HRP (Rz = 3.1). The La-HRP protein is absorbed in the plasma membrane of the plant and animal, leading to the change in the function of the cell membrane. Therefore, the La-HRP protein is harmful to living organisms.

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“…Thus, HRP produced in insect cells can be expected to have a structure and function that is similar to the native form. Indeed, HRP has been expressed in an active form in a SF9 insect cell line using a baculovirus expression systems . Silkworm has advantages over such cell culture‐based expression systems in terms of production efficiency.…”

Section: Introductionmentioning

confidence: 99%

Expression and Activation of Horseradish Peroxidase-Protein A/G Fusion Protein in Silkworm Larvae for Diagnostic Purposes

et al. 2018

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Recombinant protein production can create artificial proteins with desired functions by introducing genetic modifications to the target proteins. Horseradish peroxidase (HRP) has been used extensively as a reporter enzyme in biotechnological applications; however, recombinant production of HRP has not been very successful, hampering the utilization of HRP with genetic modifications. A fusion protein comprising an antibody binding protein and HRP will be an ideal bio-probe for high-quality HRP-based diagnostic systems. A HRP-protein A/G fusion protein (HRP-pAG) is designed and its production in silkworm (Bombyx mori) is evaluated for the first time. HRP-pAG is expressed in a soluble apo form, and is activated successfully by incubating with hemin. The activated HRP-pAG is used directly for ELISA experiments and retains its activity over 20 days at 4 °C. Moreover, HRP-pAG is modified with biotin by the microbial transglutaminase (MTG) reaction. The biotinylated HRP-pAG is conjugated with streptavidin to form a HRP-pAG multimer and the multimeric HRP-pAG produced higher signals in the ELISA system than monomeric HRP-pAG. The successful production of recombinant HRP in silkworm will contribute to creating novel HRP-based bioconjugates as well as further functionalization of HRP by applying enzymatic post-translational modifications.

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High-level expression and purification of recombinant horseradish peroxidase isozyme C in SF-9 insect cell culture

Cited by 21 publications

References 11 publications

Purification of Peroxidase from Horseradish (Armoracia rusticana) Roots

Purification of Peroxidase from Horseradish (Armoracia rusticana) Roots

The formation of a new horseradish peroxidase binding rare earth

Expression and Activation of Horseradish Peroxidase-Protein A/G Fusion Protein in Silkworm Larvae for Diagnostic Purposes

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