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      Cloning and constitutive expression of Deschampsia antarctica Cu/Zn superoxide dismutase in Pichia pastoris

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          Abstract

          Background

          Deschampsia antarctica shows tolerance to extreme environmental factors such as low temperature, high light intensity and an increasing UV radiation as result of the Antarctic ozone layer thinning. It is very likely that the survival of this species is due to the expression of genes that enable it to tolerate high levels of oxidative stress. On that account, we planned to clone the D. antarctica Cu/ZnSOD gene into Pichia pastoris and to characterize the heterologous protein.

          Findings

          The Copper/Zinc superoxide dismutase (Cu/ZnSOD) gene, SOD gene, was isolated from a D. antarctica by cDNA library screening. This SOD gene was cloned in the expression vector pGAPZαA and successfully integrated into the genome of the yeast P. pastoris SMD1168H. A constitutive expression system for the expression of the recombinant SOD protein was used. The recombinant protein was secreted into the YPD culture medium as a glycosylated protein with a 32 mg/l expression yield. The purified recombinant protein possesses a specific activity of 440 U/mg.

          Conclusion

          D. antarctica Cu/ZnSOD recombinant protein was expressed in a constitutive system, and purified in a single step by means of an affinity column. The recombinant SOD was secreted to the culture medium as a glycoprotein, corresponding to approximately 13% of the total secreted protein. The recombinant protein Cu/ZnSOD maintains 60% of its activity after incubation at 40°C for 30 minutes and it is stable (80% of activity) between -20°C and 20°C. The recombinant SOD described in this study can be used in various biotechnological applications.

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          Most cited references 27

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          Evidence for Chilling-Induced Oxidative Stress in Maize Seedlings and a Regulatory Role for Hydrogen Peroxide.

          We have taken advantage of an acclimation phenomenon in a chilling-sensitive maize inbred to investigate the molecular, biochemical, and physiological responses to chilling in preemergent maize seedlings. Three-day-old seedlings were exposed to 4[deg]C for 7 days and did not survive chilling stress unless they were preexposed to 14[deg]C for 3 days. cDNAs representing three chilling acclimation-responsive (CAR) genes were isolated by subtraction hybridization and differential screening and found to be differentially expressed during acclimation. Identification of one of these CAR genes as cat3, which encodes the mitochondrial catalase3 isozyme, led us to hypothesize that chilling imposes oxidative stress in the seedlings. Hydrogen peroxide levels were elevated during both acclimation and chilling of nonacclimated seedlings. Further molecular and biochemical analyses indicated that whereas superoxide dismutase activity was not affected, the levels of cat3 transcripts and the activities of catalase3 and guaiacol peroxidase were elevated in mesocotyls during acclimation. Accumulation of H2O2 following a short treatment with aminotriazole, a catalase inhibitor, indicated that catalase3 seems to be an important H2O2-scavenging enzyme in the seedlings. Control 3-day-old seedlings pretreated with H2O2 or menadione, a superoxide-generating compound, at 27[deg]C induced chilling tolerance. Both of these chemical treatments also increased cat3 transcripts and catalase3 and guaiacol peroxidase activities. We suggest that peroxide has dual effects at low temperatures. During acclimation, its early accumulation signals the production of antioxidant enzymes such as catalase3 and guaiacol peroxidase. At 4[deg]C, in nonacclimated seedlings, it accumulates to damaging levels in the tissues due to low levels of these, and perhaps other, antioxidant enzymes.
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            Oxidative stress: molecular perception and transduction of signals triggering antioxidant gene defenses

            Molecular oxygen (O2) is the premier biological electron acceptor that serves vital roles in fundamental cellular functions. However, with the beneficial properties of O2 comes the inadvertent formation of reactive oxygen species (ROS) such as superoxide (O2 ·- ), hydrogen peroxide, and hydroxyl radical (OH · ). If unabated, ROS pose a serious threat to or cause the death of aerobic cells. To minimize the damaging effects of ROS, aerobic organisms evolved non-enzymatic and enzymatic antioxidant defenses. The latter include catalases, peroxidases, superoxide dismutases, and glutathione S-transferases (GST). Cellular ROS-sensing mechanisms are not well understood, but a number of transcription factors that regulate the expression of antioxidant genes are well characterized in prokaryotes and in yeast. In higher eukaryotes, oxidative stress responses are more complex and modulated by several regulators. In mammalian systems, two classes of transcription factors, nuclear factor kB and activator protein-1, are involved in the oxidative stress response. Antioxidant-specific gene induction, involved in xenobiotic metabolism, is mediated by the "antioxidant responsive element" (ARE) commonly found in the promoter region of such genes. ARE is present in mammalian GST, metallothioneine-I and MnSod genes, but has not been found in plant Gst genes. However, ARE is present in the promoter region of the three maize catalase (Cat) genes. In plants, ROS have been implicated in the damaging effects of various environmental stress conditions. Many plant defense genes are activated in response to these conditions, including the three maize Cat and some of the superoxide dismutase (Sod) genes.
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              Role of antioxidants in prophylaxis and therapy: A pharmaceutical perspective.

              Antioxidants are emerging as prophylactic and therapeutic agents. These are the agents, which scavenge free radicals otherwise reactive oxygen species and prevent the damage caused by them. Free radicals have been associated with pathogenesis of various disorders like cancer, diabetes, cardiovascular diseases, autoimmune diseases, neurodegenerative disorders and are implicated in aging. Several antioxidants like SOD, CAT, epigallocatechin-3-O-gallate, lycopene, ellagic acid, coenzyme Q10, indole-3-carbinol, genistein, quercetin, vitamin C and vitamin E have been found to be pharmacologically active as prophylactic and therapeutic agents for above mentioned diseases. Antioxidants are part of diet but their bioavailability through dietary supplementation depends on several factors. This major drawback of dietary agents may be due to one or many of the several factors like poor solubility, inefficient permeability, instability due to storage of food, first pass effect and GI degradation. Conventional dosage forms may not result in efficient formulation owing to their poor biopharmaceutical properties. Principles of novel drug delivery systems need to be applied to significantly improve the performance of antioxidants. Novel drug delivery systems (NDDS) would also help in delivery of these antioxidants by oral route, as this route is of prime importance when antioxidants are intended for prophylactic purpose. Implication of NDDS for the delivery of antioxidants is largely governed by physicochemical characteristics, biopharmaceutical properties and pharmacokinetic parameters of the antioxidant to be formulated. Recently, chemical modifications, coupling agents, liposomes, microparticles, nanoparticles and gel-based systems have been explored for the delivery of these difficult to deliver molecules. Results from several studies conducted across the globe are positive and provided us with new anticipation for the improvement of human healthcare.
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                Author and article information

                Journal
                BMC Res Notes
                BMC Research Notes
                BioMed Central
                1756-0500
                2009
                12 October 2009
                : 2
                : 207
                Affiliations
                [1 ]Laboratorio de Biología Molecular Aplicada, Instituto de Agroindustrias, Facultad de Ciencias Agropecuarias y Forestales, Universidad de La Frontera, Casilla 54-D, Temuco-Chile
                [2 ]Departamento de Botánica, Facultad de Ciencias Naturales y Oceanográficas Universidad de Concepción, Casilla 160-C, Concepción-Chile
                [3 ]VentureLab, Escuela de Negocios, Universidad Adolfo Ibáñez, Av. Diagonal Las Torres 2700, Peñalolén, Santiago-Chile
                Article
                1756-0500-2-207
                10.1186/1756-0500-2-207
                2770473
                19821975
                Copyright © 2009 Gidekel et al; licensee BioMed Central Ltd.

                This is an Open Access article distributed under the terms of the Creative Commons Attribution License ( http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

                Categories
                Short Report

                Medicine

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