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dc.date.accessioned | 2021-09-24T17:52:50Z | |
dc.date.available | 2021-09-24T17:52:50Z | |
dc.date.issued | 2020-09-17 | |
dc.identifier.uri | http://sedici.unlp.edu.ar/handle/10915/125603 | |
dc.description.abstract | The yeast Pichia pastoris is a cost-effective and easily scalable system for recombinant protein production. In this work we compared the conformation of the receptor binding domain (RBD) from SARS-CoV-2 Spike protein expressed in P. pastoris and in the well established HEK-293T mammalian cell system. RBD obtained from both yeast and mammalian cells was properly folded, as indicated by UV-absorption, circular dichroism and tryptophan fluorescence. They also had similar stability, as indicated by temperature-induced unfolding (observed Tm were 50 °C and 52 °C for RBD produced in P. pastoris and HEK-293T cells, respectively). Moreover, the stability of both variants was similarly reduced when the ionic strength was increased, in agreement with a computational analysis predicting that a set of ionic interactions may stabilize RBD structure. Further characterization by HPLC, size-exclusion chromatography and mass spectrometry revealed a higher heterogeneity of RBD expressed in P. pastoris relative to that produced in HEK-293T cells, which disappeared after enzymatic removal of glycans. The production of RBD in P. pastoris was scaled-up in a bioreactor, with yields above 45 mg/L of 90% pure protein, thus potentially allowing large scale immunizations to produce neutralizing antibodies, as well as the large scale production of serological tests for SARS-CoV-2. | en |
dc.language | en | es |
dc.subject | Circular dichroism | es |
dc.subject | Antibody | es |
dc.subject | High-performance liquid chromatography | es |
dc.subject | Enzyme | es |
dc.subject | Chemistry | es |
dc.subject | Ionic strength | es |
dc.subject | Glycan | es |
dc.subject | Yeast | es |
dc.subject | Pichia pastoris | es |
dc.subject | Biochemistry | es |
dc.subject | COVID-19 | es |
dc.subject | SARS-CoV-2 | es |
dc.title | Structural and Functional Comparison of SARS-CoV-2-Spike Receptor Binding Domain Produced in Pichia pastoris and Mammalian Cells | en |
dc.type | Articulo | es |
sedici.identifier.other | doi:10.1101/2020.09.17.300335 | es |
sedici.creator.person | Gasulla, Javier | es |
sedici.creator.corporate | Argentinian AntiCovid Consortium | es |
sedici.subject.materias | Bioquímica | es |
sedici.description.fulltext | true | es |
mods.originInfo.place | Centro de Investigaciones del Medioambiente | es |
sedici.subtype | Preprint | es |
sedici.rights.license | Creative Commons Attribution 4.0 International (CC BY 4.0) | |
sedici.rights.uri | http://creativecommons.org/licenses/by/4.0/ |