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Structural and functional comparison of SARS-CoV-2-spike receptor binding domain produced in Pichia pastoris and mammalian cells.
Sci Rep ; 10(1): 21779, 2020 12 11.
Article in English | MEDLINE | ID: covidwho-970872
Preprint
This scientific journal article is probably based on a previously available preprint. It has been identified through a machine matching algorithm, human confirmation is still pending.
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Semantic information from SemMedBD (by NLM)
1. HEK293 Cells PART_OF Mammals
Subject
HEK293 Cells
Predicate
PART_OF
Object
Mammals
2. HEK293 Cells LOCATION_OF M Protei
Subject
HEK293 Cells
Predicate
LOCATION_OF
Object
M Protei
3. Extraction USES Enzymes
Subject
Extraction
Predicate
USES
Object
Enzymes
4. HEK293 Cells PART_OF Mammals
Subject
HEK293 Cells
Predicate
PART_OF
Object
Mammals
5. HEK293 Cells LOCATION_OF M Protein, multiple myeloma
Subject
HEK293 Cells
Predicate
LOCATION_OF
Object
M Protein, multiple myeloma
6. Extraction USES Enzymes
Subject
Extraction
Predicate
USES
Object
Enzymes
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 severe acute respiratory syndrome coronavirus-2 (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 high-performance liquid chromatography, 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.
Subject(s)

Full text: Available Collection: International databases Database: MEDLINE Main subject: Spike Glycoprotein, Coronavirus / SARS-CoV-2 Type of study: Prognostic study Topics: Variants Limits: Humans Language: English Journal: Sci Rep Year: 2020 Document Type: Article

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Full text: Available Collection: International databases Database: MEDLINE Main subject: Spike Glycoprotein, Coronavirus / SARS-CoV-2 Type of study: Prognostic study Topics: Variants Limits: Humans Language: English Journal: Sci Rep Year: 2020 Document Type: Article