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

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 syste...

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Detalles Bibliográficos
Autores principales: Gasulla, Javier, Argentinian AntiCovid Consortium
Formato: Articulo Preprint
Lenguaje:Inglés
Publicado: 2020
Materias:
Bioquímica
Circular dichroism
Antibody
High-performance liquid chromatography
Enzyme
Chemistry
Ionic strength
Glycan
Yeast
Pichia pastoris
Biochemistry
COVID-19
SARS-CoV-2
Acceso en línea:http://sedici.unlp.edu.ar/handle/10915/125603
Aporte de:
SEDICI (UNLP) de Universidad Nacional de La Plata
id I19-R120-10915-125603
record_format dspace
institution Universidad Nacional de La Plata
institution_str I-19
repository_str R-120
collection SEDICI (UNLP)
language Inglés
topic Bioquímica
Circular dichroism
Antibody
High-performance liquid chromatography
Enzyme
Chemistry
Ionic strength
Glycan
Yeast
Pichia pastoris
Biochemistry
COVID-19
SARS-CoV-2
spellingShingle Bioquímica
Circular dichroism
Antibody
High-performance liquid chromatography
Enzyme
Chemistry
Ionic strength
Glycan
Yeast
Pichia pastoris
Biochemistry
COVID-19
SARS-CoV-2
Gasulla, Javier
Argentinian AntiCovid Consortium
Structural and Functional Comparison of SARS-CoV-2-Spike Receptor Binding Domain Produced in Pichia pastoris and Mammalian Cells
topic_facet Bioquímica
Circular dichroism
Antibody
High-performance liquid chromatography
Enzyme
Chemistry
Ionic strength
Glycan
Yeast
Pichia pastoris
Biochemistry
COVID-19
SARS-CoV-2
description 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.
format Articulo
Preprint
author Gasulla, Javier
Argentinian AntiCovid Consortium
author_facet Gasulla, Javier
Argentinian AntiCovid Consortium
author_sort Gasulla, Javier
title Structural and Functional Comparison of SARS-CoV-2-Spike Receptor Binding Domain Produced in Pichia pastoris and Mammalian Cells
title_short Structural and Functional Comparison of SARS-CoV-2-Spike Receptor Binding Domain Produced in Pichia pastoris and Mammalian Cells
title_full Structural and Functional Comparison of SARS-CoV-2-Spike Receptor Binding Domain Produced in Pichia pastoris and Mammalian Cells
title_fullStr Structural and Functional Comparison of SARS-CoV-2-Spike Receptor Binding Domain Produced in Pichia pastoris and Mammalian Cells
title_full_unstemmed Structural and Functional Comparison of SARS-CoV-2-Spike Receptor Binding Domain Produced in Pichia pastoris and Mammalian Cells
title_sort structural and functional comparison of sars-cov-2-spike receptor binding domain produced in pichia pastoris and mammalian cells
publishDate 2020
url http://sedici.unlp.edu.ar/handle/10915/125603
work_keys_str_mv AT gasullajavier structuralandfunctionalcomparisonofsarscov2spikereceptorbindingdomainproducedinpichiapastorisandmammaliancells
AT argentiniananticovidconsortium structuralandfunctionalcomparisonofsarscov2spikereceptorbindingdomainproducedinpichiapastorisandmammaliancells
bdutipo_str Repositorios
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