RT Journal Article
SR Electronic
T1 The nuts and bolts of SARS-CoV-2 Spike Receptor Binding Domain heterologous expression
JF bioRxiv
FD Cold Spring Harbor Laboratory
SP 2021.09.17.460782
DO 10.1101/2021.09.17.460782
A1 Mariano Maffei
A1 Linda Celeste Montemiglio
A1 Grazia Vitagliano
A1 Luigi Fedele
A1 Shaila Sellathurai
A1 Federica Bucci
A1 Mirco Compagnone
A1 Valerio Chiarini
A1 Cécile Exertier
A1 Alessia Muzi
A1 Giuseppe Roscilli
A1 Beatrice Vallone
A1 Emanuele Marra
YR 2021
UL http://biorxiv.org/content/early/2021/09/17/2021.09.17.460782.abstract
AB COVID-19 is a highly infectious disease caused by a newly emerged coronavirus (SARS-CoV-2) that has rapidly progressed into a pandemic. This unprecedent emergency has stressed the significance of developing effective therapeutics to fight current and future outbreaks. The receptor-binding domain (RBD) of the SARS-CoV-2 surface Spike protein is the main target for vaccines and represents a helpful “tool” to produce neutralizing antibodies or diagnostic kits. In this work, we provide a detailed characterization of the native RBD produced in three major model systems: Escherichia coli, insect and HEK-293 cells. Circular dichroism, gel filtration chromatography and thermal denaturation experiments indicated that recombinant SARS-CoV-2 RBD proteins are stable and correctly folded. In addition, their functionality and receptor-binding ability were further evaluated through ELISA, flow cytometry assays and bio-layer interferometry.Competing Interest StatementMariano Maffei, Grazia Vitagliano, Shaila Sellathurai, Federica Bucci, Alessia Muzi and Valerio Chiarini are employees of Takis and Evvivax companies. Mirco Compagnone is employee of NeoMatrix. Luigi Fedele is a former employee of Takis company. Giuseppe Roscilli and Emanuele Marra are co-founders of Takis and Evvivax companies.ACE2angiotensin-converting enzyme 2BLIbio-layer interferometryCDcircular dichroismCOVID-19coronavirus disease-2019ELISAenzyme-linked immunosorbent assayFACSfluorescence-activated cell sortingHEKhuman embryonic kidneyIgGimmunoglobulin GIMACimmobilized metal affinity chromatographyMALDI-MSmatrix-assisted laser desorption/ionization- mass spectrometryMFImean fluorescence intensity/ geometric meanMSmass spectrometryMWmolecular weightMWCOmolecular weight cut-offODoptical densityPMFpeptide mass fingerprintPMTVphotomultiplier voltagePOIprotein of interestRBDreceptor binding domainE. coli-RBDreceptor binding domain produced in E. coliHEK-293-RBDreceptor binding domain produced in HEK293 cellsinsect-RBDreceptor binding domain produced in insect cellsRP-UHPLC-MSreversed-phase - ultra-high performance liquid chromatography - mass spectrometrySARS-CoV-2severe acute respiratory syndrome coronavirus 2SECsize exclusion chromatographyTmmelting temperatureUHPLCultra-high performance liquid chromatographyWHOWorld Health Organization