Druggability and Genetic Variability of the ADP-bound Pocket of SARS-CoV-2 RNA-dependent RNA polymerase NiRAN domain Across Coronaviruses and SARS-CoV-2 Samples – Post 22

Posted onAuthorSetayesh YazdaniLeave a comment

The RNA-dependent RNA-polymerase (RdRp) also known as non-structural protein 12 (nsp12) is the target of antiviral agent remdesivir. Nsp12 has an important role in viral genome replication and transcription. (Chen et al., 2020)  Chen et al. identified a new pocket on the N-terminal extension of nsp12 occupied by ADP-Mg2+ after solving the structure of the helicase-polymerase complex. This ADP-bound pocket is on the N-terminal nidovirus RdRp-associated nucleotidyltransferase (NiRAN) domain which Read More …

Interrogating PR/SET domains for the ability to bind SAH

Posted onAuthorDanton IvanochkoLeave a comment

This is an excerpt from my PhD thesis where I investigated 13 human PR/SET domains from the PRDM protein family and assessed their ability to bind to a fluorinated SAH analog. The research helps to identify which of the PRDMs could be methyltransferases. This work was carried out in the laboratory of Dr. Cheryl Arrowsmith Read More …

Production and purification of Ectodomain of SARS-CoV-2 Spike (S) protein

Posted onAuthorAlma SeitovaLeave a comment

Production and purification of Ectodomain of SARS-CoV-2 Spike (S) protein Two different expression plasmids for prefusion S ectodomain residues 1−1208 of 2019-nCoV S (GenBank: MN908947) with proline substitutions (2P) at residues 986 and 987(1) and with six proline substitutions (HexaPro (at residues 817, 892, 899, 942, 986 and 987 (2), a “GSAS” substitution at the Read More …

Development and Optimization of an IQF USP5 Activity Assay

Posted onAuthorMandeep MannLeave a comment

In a previous post, I described a ubiquitin-rhodamine110 (UbRho110) assay, and showed that a ZnF-UBD ligand antagonizes USP5 catalytic activity. UbRho110 is not a native substrate, and validation with native polyubiquitin species in an orthogonal assay is required. To address this, I developed and optimized an internally quenched fluorophore pair (IQF) assay. You can see Read More …

Genetic Variability at An Allosteric site of SARS-CoV-2 RNA-dependent RNA polymerase Across Coronaviruses and SARS-CoV-2 Samples – Post 21

Posted onAuthorSetayesh YazdaniLeave a comment

SARS-CoV-2 is a positive-strand RNA virus, depending on its multi-subunit machinery to replicate its RNA. This machinery is known as RNA-dependent RNA polymerase (RdRp). The catalytic subunit of RdRp, which is the core component of this machinery, is called nsp12. Nsp12 has little catalytic activity on its own and relies on accessory subunits to have Read More …

FBXW11 interaction with β-catenin – cellular assay

Posted onAuthorMagdalena SzewczykLeave a comment

Background The SCF (SKP1-CUL1-F-box protein) E3 ubiquitin ligase is a complex of proteins that helps facilitate the ubiquitination of substrates for targeted proteasomal degradation. FBXW11 is F-box containing protein that has 11 WD-repeats and is a substrate recognition component of the SCF complex. FBXW11 recognizes and binds its substrates, including beta-catenin, at phosphorylated sites or Read More …

FBXW7 interaction with Cyclin E1 – cellular assay

Posted onAuthorMagdalena Szewczyk1 Comment

Background The SCF (SKP1-CUL1-F-box protein) E3 ubiquitin ligase is a complex of proteins that helps facilitate the ubiquitination of substrates for targeted proteasomal degradation. FBXW7 (F-box/WD repeat-containing protein 7) is an F-box protein component of the SCF complex and is involved in substrate recognition. The F-box domain tethers FBXW7 to the SCF complex by interacting Read More …

Production and purification of recombinant SARS COV-2 RBDSD1-Fc protein

Posted onAuthorAlma SeitovaLeave a comment

Production and purification of recombinant SARS COV-2 RBDSD1-Fc protein from a transiently transfected EXPI293 mammalian cells. Expression plasmid for CoV-9-RBDSD1, residues of RBD R319−S591 with a C-terminal HRV3C protease cleavage site, a monomeric Fc tag and an 8XHisTag was generously gifted by Dr. McLellan’s lab (1). Secreted proteins were purified from a suspension culture cell Read More …

Production and purifications of the SARS-CoV-2 related proteins from the transiently transfected mammalian cells

Posted onAuthorAlma SeitovaLeave a comment

Over the last few months, we have all become familiar with the COVID-19 global pandemic. The coronavirus is “decorated” with the Spike (S) protein, one of the 25 proteins of SARS-CoV-2 that are encoded in the viral genome. The infection starts with the attachment of the virus to the cell membrane to enter host cells. Read More …

Genetic Variability at the Remdesivir-binding Pocket of SARS-CoV-2 RNA-dependent RNA polymerase Across Coronaviruses and SARS-CoV-2 Samples – Post 20

Posted onAuthorSetayesh YazdaniLeave a comment

In my previous post 19, I showed how we assessed the druggability of the remdesivir-binding site of SARS-CoV-2 RNA-dependent RNA polymerase (RdRp) and determined the residues lining that site. In this post, we analyze the genetic diversity of RdRp catalytic site across Alpha- and Betacoronavirus entries from UniProt and among SARS-CoV-2 samples. In the context Read More …