Abstract
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 Statement
Mariano 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.
Abbreviations
- ACE2
- angiotensin-converting enzyme 2
- BLI
- bio-layer interferometry
- CD
- circular dichroism
- COVID-19
- coronavirus disease-2019
- ELISA
- enzyme-linked immunosorbent assay
- FACS
- fluorescence-activated cell sorting
- HEK
- human embryonic kidney
- IgG
- immunoglobulin G
- IMAC
- immobilized metal affinity chromatography
- MALDI-MS
- matrix-assisted laser desorption/ionization- mass spectrometry
- MFI
- mean fluorescence intensity/ geometric mean
- MS
- mass spectrometry
- MW
- molecular weight
- MWCO
- molecular weight cut-off
- OD
- optical density
- PMF
- peptide mass fingerprint
- PMTV
- photomultiplier voltage
- POI
- protein of interest
- RBD
- receptor binding domain
- E. coli-RBD
- receptor binding domain produced in E. coli
- HEK-293-RBD
- receptor binding domain produced in HEK293 cells
- insect-RBD
- receptor binding domain produced in insect cells
- RP-UHPLC-MS
- reversed-phase - ultra-high performance liquid chromatography - mass spectrometry
- SARS-CoV-2
- severe acute respiratory syndrome coronavirus 2
- SEC
- size exclusion chromatography
- Tm
- melting temperature
- UHPLC
- ultra-high performance liquid chromatography
- WHO
- World Health Organization
