Cloning and constitutive expression of Deschampsia antarcticaCu/Zn superoxide dismutase in Pichia pastoris
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 ge...
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ftbiomed:oai:biomedcentral.com:1756-0500-2-207 2023-05-15T13:36:40+02:00 Cloning and constitutive expression of Deschampsia antarcticaCu/Zn superoxide dismutase in Pichia pastoris Sánchez-Venegas, Jaime R Navarrete, Alejandro Dinamarca, Jorge Bravo Ramírez, León A Moraga, Ana Gidekel, Manuel 2009-10-12 http://www.biomedcentral.com/1756-0500/2/207 en eng BioMed Central Ltd. http://www.biomedcentral.com/1756-0500/2/207 Copyright 2009 Gidekel et al; licensee BioMed Central Ltd. Short Report 2009 ftbiomed 2009-11-08T00:22:50Z 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. Other/Unknown Material Antarc* Antarctic Antarctica BioMed Central Antarctic The Antarctic |
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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. |
format |
Other/Unknown Material |
author |
Sánchez-Venegas, Jaime R Navarrete, Alejandro Dinamarca, Jorge Bravo Ramírez, León A Moraga, Ana Gidekel, Manuel |
spellingShingle |
Sánchez-Venegas, Jaime R Navarrete, Alejandro Dinamarca, Jorge Bravo Ramírez, León A Moraga, Ana Gidekel, Manuel Cloning and constitutive expression of Deschampsia antarcticaCu/Zn superoxide dismutase in Pichia pastoris |
author_facet |
Sánchez-Venegas, Jaime R Navarrete, Alejandro Dinamarca, Jorge Bravo Ramírez, León A Moraga, Ana Gidekel, Manuel |
author_sort |
Sánchez-Venegas, Jaime R |
title |
Cloning and constitutive expression of Deschampsia antarcticaCu/Zn superoxide dismutase in Pichia pastoris |
title_short |
Cloning and constitutive expression of Deschampsia antarcticaCu/Zn superoxide dismutase in Pichia pastoris |
title_full |
Cloning and constitutive expression of Deschampsia antarcticaCu/Zn superoxide dismutase in Pichia pastoris |
title_fullStr |
Cloning and constitutive expression of Deschampsia antarcticaCu/Zn superoxide dismutase in Pichia pastoris |
title_full_unstemmed |
Cloning and constitutive expression of Deschampsia antarcticaCu/Zn superoxide dismutase in Pichia pastoris |
title_sort |
cloning and constitutive expression of deschampsia antarcticacu/zn superoxide dismutase in pichia pastoris |
publisher |
BioMed Central Ltd. |
publishDate |
2009 |
url |
http://www.biomedcentral.com/1756-0500/2/207 |
geographic |
Antarctic The Antarctic |
geographic_facet |
Antarctic The Antarctic |
genre |
Antarc* Antarctic Antarctica |
genre_facet |
Antarc* Antarctic Antarctica |
op_relation |
http://www.biomedcentral.com/1756-0500/2/207 |
op_rights |
Copyright 2009 Gidekel et al; licensee BioMed Central Ltd. |
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1766082301038428160 |