Browse
Submit Data
Databases
API
Help

Dataset Information

35 Views

0 Connections

0 Citations

0 Reanalyses

0 Downloads

Omics score: 0

Functional Trimeric SARS-CoV-2 Envelope Protein Expressed in Stable CHO Cells.

ABSTRACT: The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), a β-coronavirus, is the causative agent of the COVID-19 pandemic. One of the three membrane-bound envelope proteins is the spike protein (S), the one responsible for docking to the cellular surface protein ACE2 enabling infection with SARS-CoV-2. Although the structure of the S-protein has distinct similarities to other viral envelope proteins, robust and straightforward protocols for recombinant expression and purification are not described in the literature. Therefore, most studies are done with truncated versions of the protein, like the receptor-binding domain. To learn more about the interaction of the virus with the ACE2 and other cell surface proteins, it is mandatory to provide recombinant spike protein in high structural quality and adequate quantity. Additional mutant variants will give new insights on virus assembly, infection mechanism, and therapeutic drug development. Here, we describe the development of a recombinant CHO cell line stably expressing the extracellular domain of a trimeric variant of the SARS CoV-2 spike protein and discuss significant parameters to be considered during the expression and purification process.

SUBMITTER: Mayrhofer P

PROVIDER: S-EPMC8718689 | biostudies-literature |

REPOSITORIES: biostudies-literature

ACCESS DATA

Json Xml

Similar Datasets

Antigenicity and immunogenicity of SARS-CoV S protein receptor-binding domain stably expressed in CHO cells.

Project description:The receptor-binding domain (RBD) of SARS coronavirus (SARS-CoV) spike (S) protein contains multiple conformation-dependent epitopes that induce neutralizing antibody responses. Here we used CHO-K1 cells to establish a cell line for stable expression of a 193-mer (residues 318-510) RBD (RBD193-CHO) and determined its antigenicity and immunogenicity. We found that RBD193-CHO reacted strongly with a panel of six monoclonal antibodies recognizing various conformational and linear epitopes in RBD, suggesting that this recombinant protein maintains intact conformation and good antigenicity. Immunization of mice with RBD193-CHO resulted in induction of high titers of RBD-specific neutralizing antibodies and potent IL-4-expressing T cell responses. RBD193-CHO induced immunity that protected a majority of the vaccinated mice from SARS-CoV challenge. These results suggest that the recombinant RBD produced in an established stable cell line maintains strong immunogenicity with high potential for use as an effective and economic subunit SARS vaccine.

| S-EPMC2750803 | biostudies-literature

A Uniquely Stable Trimeric Model of SARS-CoV-2 Spike Transmembrane Domain.

Project description:Understanding fusion mechanisms employed by SARS-CoV-2 spike protein entails realistic transmembrane domain (TMD) models, while no reliable approaches towards predicting the 3D structure of transmembrane (TM) trimers exist. Here, we propose a comprehensive computational framework to model the spike TMD only based on its primary structure. We performed amino acid sequence pattern matching and compared the molecular hydrophobicity potential (MHP) distribution on the helix surface against TM homotrimers with known 3D structures and selected an appropriate template for homology modeling. We then iteratively built a model of spike TMD, adjusting "dynamic MHP portraits" and residue variability motifs. The stability of this model, with and without palmitoyl modifications downstream of the TMD, and several alternative configurations (including a recent NMR structure), was tested in all-atom molecular dynamics simulations in a POPC bilayer mimicking the viral envelope. Our model demonstrated unique stability under the conditions applied and conforms to known basic principles of TM helix packing. The original computational framework looks promising and could potentially be employed in the construction of 3D models of TM trimers for a wide range of membrane proteins.

| S-EPMC9409440 | biostudies-literature

Delivery of recombinant SARS-CoV-2 envelope protein into human cells.

Project description:SARS-CoV-2 envelope protein (S2-E) is a conserved membrane protein that is essential to coronavirus assembly and budding. Here, we describe the recombinant expression and purification of S2-E into amphipol-class amphipathic polymer solutions. The physical properties of amphipols underpin their ability to solubilize and stabilize membrane proteins without disrupting membranes. Amphipol delivery of S2-E to pre-formed planar bilayers results in spontaneous membrane integration and formation of viroporin ion channels. Amphipol delivery of the S2-E protein to human cells results in membrane integration followed by retrograde trafficking to a location adjacent to the endoplasmic reticulum-to-Golgi intermediate compartment (ERGIC) and the Golgi, which are the sites of coronavirus replication. Delivery of S2-E to cells enables both chemical biological approaches for future studies of SARS-CoV-2 pathogenesis and development of "Trojan Horse" anti-viral therapies. This work also establishes a paradigm for amphipol-mediated delivery of membrane proteins to cells.

| S-EPMC7899446 | biostudies-literature

CryoEM SPA dataset for NB21, SARS-Cov-2 and NB105 trimeric complex

Project description: Not available

| EMPIAR-10837 | biostudies-other

CryoEM SPA dataset for NB21, SARS-Cov-2 and NB36 trimeric complex

Project description: Not available

| EMPIAR-10838 | biostudies-other

The PDZ-binding motif of SARS-CoV envelope protein is a determinant of viral pathogenesis

Project description:A recombinant SARS-CoV lacking the envelope (E) protein is attenuated in vivo. Here we report that E protein PDZ-binding motif (PBM), a domain involved in protein-protein interactions, is a major virulence determinant in vivo. Elimination of SARS-CoV E protein PBM by using reverse genetics led to attenuated viruses (SARS-CoV-mutPBM) and to a reduction in the deleterious exacerbate immune response triggered during infection with the parental virus (SARS-CoV-wt). Cellular protein syntenin bound E protein PBM during SARS-CoV infection. Syntenin activates p38 MAPK leading to overexpression of inflammatory cytokines, and we have shown that active p38 MAPK was reduced in lungs of mice infected with SARS-CoVs lacking E protein PBM (SARS-CoV-mutPBM) as compared with the parental virus (SARS-CoV-wt), leading to a decreased expression of inflammatory cytokines and to viral attenuation. Therefore, E protein PBM is a virulence factor that activates pathogenic immune response most likely by using syntenin as a mediator of p38 MAPK induced inflammation. Three biological replicates were independently hybridized (one channel per slide) for each sample type (SARS-CoV-wt, SARS-CoV-mutPBM, Mock). Slides were Sure Print G3 Agilent 8x60K Mouse (G4852A-028005)

2014-10-31 | E-GEOD-52920 | biostudies-arrayexpress

SARS-CoV-2 envelope protein topology in eukaryotic membranes.

Project description:Coronavirus E protein is a small membrane protein found in the virus envelope. Different coronavirus E proteins share striking biochemical and functional similarities, but sequence conservation is limited. In this report, we studied the E protein topology from the new SARS-CoV-2 virus both in microsomal membranes and in mammalian cells. Experimental data reveal that E protein is a single-spanning membrane protein with the N-terminus being translocated across the membrane, while the C-terminus is exposed to the cytoplasmic side (Ntlum/Ctcyt). The defined membrane protein topology of SARS-CoV-2 E protein may provide a useful framework to understand its interaction with other viral and host components and contribute to establish the basis to tackle the pathogenesis of SARS-CoV-2.

| S-EPMC7536074 | biostudies-literature

Mutational insights into the envelope protein of SARS-CoV-2.

Project description:The ongoing mutations in the structural proteins of SARS-CoV-2 are the major impediment for prevention and control of the COVID-19 disease. Presently we focused on evolution of the envelope (E) protein, one of the most enigmatic and less studied protein among the four structural proteins (S, E, M and N) associated with multitude of immunopathological functions of SARS-CoV-2. In the present study, we comprehensively analyzed 81,818 high quality E protein sequences of SARS-CoV-2 globally available in the GISAID database as of 20 August 2020. Compared to Wuhan reference strain, our mutational analysis explored only 1.2 % (982/81818) mutant strains undergoing a total of 115 unique amino acid (aa) substitutions in the E protein, highlighting the fact that most (98.8 %) of the E protein of SARS-CoV-2 strains are highly conserved. Moreover, we found 58.77 % (134 of 228) nucleotides (nt) positions of SARS-CoV-2 E gene encountering a total of 176 unique nt-level mutations globally, which may affect the efficacy of real time RT-PCR-based molecular detection of COVID-19. Importantly, higher aa variations observed in the C-terminal domain (CTD) of the E protein, particularly at Ser55-Phe56, Arg69 and the C-terminal end (DLLV: 72-75) may alter the binding of SARS-CoV-2 Envelope protein to tight junction-associated PALS1 and thus could play a key role in COVID-19 pathogenesis. Furthermore, this study revealed the V25A mutation in the transmembrane domain which is a key factor for the homopentameric conformation of E protein. Our analysis also observed a triple cysteine motif harboring mutation (L39M, A41S, A41V, C43F, C43R, C43S, C44Y, N45R) which may hinder the binding of E protein with spike glycoprotein. These results therefore suggest the continuous monitoring of the structural proteins including the envelope protein of SARS-CoV-2 since the number of genome sequences from across the world are continuously increasing.

| S-EPMC7723457 | biostudies-literature

Effects of envelope- or membrane-protein segments of SARS-CoV-2 on gene expression of HUVEC cells

Project description:Exterior segments of E-proteins bound onto and modulated gene expression in human vascular endothelial cells in vitro The finding that EC38 segment laid out of SARS-CoV-2 virus envelope prompted us to wonder if this segment would bind onto and impact the biology of host cells. Bearing the viremia during severe COVID-19 in mind, more attention was paid to the circulation system. Cultured human umbilical vein endothelial cells (HUVECs) were used to test the binding of EC38 and MN19 peptides and the potential consequence in terms of gene expression upon such binding. To check if the binding of interest peptides onto HUVEC concurred functional consequences in the cells, HUVECs were treated with EC38 and MN19 peptides (5μM) for 24 hours, and their transcriptome was measured via RNA sequencing.

2024-07-01 | GSE268369 | GEO

Accelerated induction of apoptosis in insect cells by baculovirus-expressed SARS-CoV membrane protein.

Project description:It has been shown that severe acute respiratory syndrome-associated coronavirus (SARS-CoV) 3a and 7a proteins, but not membrane (M) protein, induce apoptosis in mammalian cells. Upon expression of SARS-CoV M protein using the baculovirus/insect cell expression system, however, we found that the expressed M protein triggered accelerated apoptosis in insect cells, as characterized by rapid cell death, elevated cytotoxicity, cell shrinkage, nuclear condensation and DNA fragmentation. Conversely, the M protein expressed in mammalian cells did not induce apoptosis. This is the first report describing the induction of apoptosis by SARS-CoV M protein in animal cells and possible implications are discussed.

| S-EPMC7094299 | biostudies-literature

OmicsDI is part of the ELIXIR infrastructure

OmicsDI is an Elixir interoperability service. Learn more ›

Tweets

OmicsDI Databases

PRIDE
PeptideAtlas
MassIVE
JPOST Repository
Physiome Model Repository

EGA
EVA
ENA
LINCS
PAXDB
Cell Collective

MetaboLights
Metabolomics Workbench
MetabolomeExpress
GNPS
BioModels
FAIRDOMHub

ArrayExpress
dbGaP
ExpressionAtlas
GEO
NODE

Information

Databases
Help
API
Contact us
Code on GitHub
Terms of use
Submit Data