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Artículo

Structural and Functional Comparison of SARS-CoV-2-Spike Receptor Binding Domain Produced in Pichia pastoris and Mammalian Cells

Zelada, Alicia MercedesIcon ; Auge, Gabriela AlejandraIcon ; Blaustein, Matías; Bredeston, Luis MaríaIcon ; Corapi, Enrique SebastianIcon ; Craig, Patricio OliverIcon ; Cossio, Leandro Andres; Dain, Liliana BeatrizIcon ; D’Alessio, Cecilia; Elias, Fernanda; Fernández, Natalia BrendaIcon ; Gasulla, JavierIcon ; Gorojovsky, NataliaIcon ; Gudesblat, Gustavo EduardoIcon ; Herrera, Maria GeorginaIcon ; Ibañez, Lorena ItatíIcon ; Idrovo Hidalgo, Tommy; Iglesias Randon, Matías; Kamenetzky, LauraIcon ; Nadra, Alejandro DanielIcon ; Noseda, Diego GabrielIcon ; Pavan, Carlos HumbertoIcon ; Pavan, Maria FlorenciaIcon ; Pignataro, María FlorenciaIcon ; Roman, Ernesto AndresIcon ; Ruberto, Lucas Adolfo MauroIcon ; Rubinstein, NataliaIcon ; Santos, JavierIcon ; Velázquez Duarte, FranciscoIcon ; Zelada, Alicia MercedesIcon
Fecha de publicación: 09/2020
Editorial: Cold Spring Harbor Laboratory Press
Revista: BioRxiv
ISSN: 0362-4331
Idioma: Inglés
Tipo de recurso: Artículo publicado
Clasificación temática:
Bioquímica y Biología Molecular

Resumen

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.
Palabras clave: SARS-CoV-2 , Pichia pastoris , HEK-293 , antigen , COVID-19
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info:eu-repo/semantics/openAccess Excepto donde se diga explícitamente, este item se publica bajo la siguiente descripción: Creative Commons Attribution-NonCommercial-ShareAlike 2.5 Unported (CC BY-NC-SA 2.5)
Identificadores
URI: http://hdl.handle.net/11336/119371
URL: https://www.biorxiv.org/content/10.1101/2020.09.17.300335v1.full
DOI: https://doi.org/10.1101/2020.09.17.300335
Colecciones
Articulos(IBBEA)
Articulos de INSTITUTO DE BIODIVERSIDAD Y BIOLOGIA EXPERIMENTAL Y APLICADA
Articulos(IMPAM)
Articulos de INSTITUTO DE INVESTIGACIONES EN MICROBIOLOGIA Y PARASITOLOGIA MEDICA
Articulos(IQUIBICEN)
Articulos de INSTITUTO DE QUIMICA BIOLOGICA DE LA FACULTAD DE CS. EXACTAS Y NATURALES
Articulos(IQUIFIB)
Articulos de INST.DE QUIMICA Y FISICO-QUIMICA BIOLOGICAS "PROF. ALEJANDRO C. PALADINI"
Articulos(NANOBIOTEC)
Articulos de INSTITUTO DE NANOBIOTECNOLOGIA
Citación
Zelada, Alicia Mercedes; Auge, Gabriela Alejandra; Blaustein, Matías; Bredeston, Luis María; Corapi, Enrique Sebastian; et al.; Structural and Functional Comparison of SARS-CoV-2-Spike Receptor Binding Domain Produced in Pichia pastoris and Mammalian Cells; Cold Spring Harbor Laboratory Press; BioRxiv; 9-2020
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