Protocol for production and purification of SARS-CoV-2 3CLpro.

3CLpro protease from SARS-CoV-2 is a primary target for COVID-19 antiviral drug development. Here, we present a protocol for 3CLpro production in Escherichia coli. We describe steps to purify 3CLpro, expressed as a fusion with the Saccharomyces cerevisiae SUMO protein, with yields up to 120 mg L-1 following cleavage. The protocol also provides isotope-enriched samples suitable for nuclear magnetic resonance (NMR) studies. We also present methods to characterize 3CLpro by mass spectrometry, X-ray crystallography, heteronuclear NMR, and a Förster-resonance-energy-transfer-based enzyme assay. For complete details on the use and execution of this protocol, please refer to Bafna et al.1.

Studying the Effect of Glycosylation towards SARS-CoV-2 Spike RBD and ACE2 Interaction by Native Mass Spectrometry

The coronavirus disease 2019 (COVID-19) pandemic caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has serious consequences on global public health and social development. The binding of receptor binding domain (RBD) of spike protein to angiotensin converting enzyme 2 (ACE2) on the surface of SARS-CoV-2 host cell initiates the infection progress. Spike and ACE2 are both glycoproteins, the impact of glycosylation on protein structures and protein-protein interactions remains largely elusive. Characterizing the structural and dynamics of protein-protein binding progress will improve mechanism understanding of viral infection and facilitate targeted drug design. Structural mass spectrometry (MS) method is widely used in protein structural studies, providing complementary information to conventional biophysical methods, such as X-ray crystallography, nuclear magnetic resonance (NMR) spectroscopy and cryo-electron microscopy (cryo-EM). Native mass spectrometry (native MS) is an emerging technology that enables the study of intact protein, non-covalent protein-protein, and protein-ligand complexes in their biological state, which can provide structural stability, binding stoichiometry, and spatial arrangement information. Here, native MS was used to examine the interaction between RBD and ACE2 as well as the impact of deglycosylation on the interaction stability of the RBD-ACE2 complex. The results revealed that both RBD and ACE2 are highly glycosylated, ACE2 presents as a dimer while RBD as a monomer, and they form a (RBD-ACE2)2 complex. The conditions of using PNGasc F to remove the N-glycan were optimized. At least two Oglycans including NcuAc(2) and GalNAcC 1) Gal( 1) NcuAc(2) or GlcNAcd ) Gal(l) NeuAc(2) were observed for the N-glycan removed RBD. Furthermore, the stability of the complexes formed by glycosylated and deglycosylated RBD with ACE2 was compared, and the results showed that the removal of N-glycan significantly drops the interaction stability of the RBD-ACE2 complex. Therefore, we recommend that glycosyla-tion should not be removed for structural and functional studies. Additional glycosyla-tion, structural and dynamics studies on Spike (including separated RBD) and ACE2 complexes would help us to understand the process of viral infection, advance drug design and vaccine developments. Nowadays, a comprehensive MS-based toolbox has been developed for the analysis of protein structure, function, and dynamics, including hydrogen-deuterium exchange MS (HDX-MS), native top-down (nTD) MS, cross-linking MS (XL-MS), and covalent labelling MS (CL-MS), etc. Through integrating structural MS methods, more detailed and comprehensive structural information about glycoproteins and their complexes will be uncovered. © 2022 Chinese Society for Mass Spectrometry. All rights reserved.

Studying the Effect of Glycosylation towards SARS-CoV-2 Spike RBD and ACE2 Interaction by Native Mass Spectrometry

The coronavirus disease 2019 (COVID-19) pandemic caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has serious consequences on global public health and social development. The binding of receptor binding domain (RBD) of spike protein to angiotensin converting enzyme 2 (ACE2) on the surface of SARS-CoV-2 host cell initiates the infection progress. Spike and ACE2 are both glycoproteins, the impact of glycosylation on protein structures and protein-protein interactions remains largely elusive. Characterizing the structural and dynamics of protein-protein binding progress will improve mechanism understanding of viral infection and facilitate targeted drug design. Structural mass spectrometry (MS) method is widely used in protein structural studies, providing complementary information to conventional biophysical methods, such as X-ray crystallography, nuclear magnetic resonance (NMR) spectroscopy and cryo-electron microscopy (cryo-EM). Native mass spectrometry (native MS) is an emerging technology that enables the study of intact protein, non-covalent protein-protein, and protein-ligand complexes in their biological state, which can provide structural stability, binding stoichiometry, and spatial arrangement information. Here, native MS was used to examine the interaction between RBD and ACE2 as well as the impact of deglycosylation on the interaction stability of the RBD-ACE2 complex. The results revealed that both RBD and ACE2 are highly glycosylated, ACE2 presents as a dimer while RBD as a monomer, and they form a (RBD-ACE2)2 complex. The conditions of using PNGasc F to remove the N-glycan were optimized. At least two Oglycans including NcuAc(2) and GalNAcC 1) Gal( 1) NcuAc(2) or GlcNAcd ) Gal(l) NeuAc(2) were observed for the N-glycan removed RBD. Furthermore, the stability of the complexes formed by glycosylated and deglycosylated RBD with ACE2 was compared, and the results showed that the removal of N-glycan significantly drops the interaction stability of the RBD-ACE2 complex. Therefore, we recommend that glycosyla-tion should not be removed for structural and functional studies. Additional glycosyla-tion, structural and dynamics studies on Spike (including separated RBD) and ACE2 complexes would help us to understand the process of viral infection, advance drug design and vaccine developments. Nowadays, a comprehensive MS-based toolbox has been developed for the analysis of protein structure, function, and dynamics, including hydrogen-deuterium exchange MS (HDX-MS), native top-down (nTD) MS, cross-linking MS (XL-MS), and covalent labelling MS (CL-MS), etc. Through integrating structural MS methods, more detailed and comprehensive structural information about glycoproteins and their complexes will be uncovered. © 2022 Chinese Society for Mass Spectrometry. All rights reserved.

Protocol to determine antibody affinity and concentration in complex solutions using microfluidic antibody affinity profiling.

Conventional methods of measuring affinity are limited by artificial immobilization, large sample volumes, and homogeneous solutions. This protocol describes microfluidic antibody affinity profiling on complex human samples in solution to obtain a fingerprint reflecting both affinity and active concentration of the target protein. To illustrate the protocol, we analyze the antibody response in SARS-CoV-2 omicron-naïve samples against different SARS-CoV-2 variants of concern. However, the protocol and the technology are amenable to a broad spectrum of biomedical questions. For complete details on the use and execution of this protocol, please refer to Emmenegger et al. (2022),1 Schneider et al. (2022),2 and Fiedler et al. (2022).3.

Inhibition of SARS-CoV-2 infection in cellular systems using engineered trimeric receptor-binding domain of spike protein.

Here, we provide a protocol for the design, expression, purification, and functional studies of an engineered trimeric version of the receptor-binding domain (tRBD) of SARS-CoV-2 spike protein. We describe the use of tRBD to block SARS-CoV-2 spike pseudovirus and true virus binding to cellular angiotensin converting enzyme-2 (ACE2), thereby blocking viral infection. This protocol is applicable to generate a trimeric version of any protein of interest. For complete details on the use and execution of this protocol, please refer to Basavarajappa et al. (2022).1.

Protocol for high-throughput screening of SARS-CoV-2 main protease inhibitors using a robust fluorescence polarization assay.

Discovery of efficacious antiviral agents targeting SARS-CoV-2 main protease (Mpro) is of the highest importance to fight against COVID-19. Here, we describe a simple protocol for high-throughput screening of Mpro inhibitors using a robust fluorescence polarization (FP) assay. Candidate Mpro inhibitors from large compound libraries could be rapidly identified by monitoring the change of millipolarization unit value. This affordable FP assay can be modified to screen antiviral agents targeting virus protease. For complete details on the use and execution of this protocol, please refer to Li et al. (2022), Yan et al. (2021), and Yan et al. (2022c).

Characterization of multiple interactions between the envelope E protein of SARS-CoV-2 and human BRD4.

The SARS-CoV-2 envelope (E) protein hijacks human BRD4 (bromodomain and extra-terminal domain protein 4). Here, we describe a protocol to characterize the interaction of the acetylated E protein with BRD4 in vivo. We detail steps to use NMR spectroscopy to map the binding interface and include steps to monitor the effect of BRD4 inhibitors in SARS-CoV-2-infected human lung bronchial epithelial cells. This approach could be applied to study interactions involving other viral and human proteins. For complete details on the use and execution of this protocol, please refer to Vann et al. (2022).1.

Transient transfection and purification of SARS-CoV-2 spike protein from mammalian cells.

SARS-CoV-2 spike (S) protein ectodomain purification can be challenging, with engineered and natural variations often resulting in lower yields. Here, we present a detailed transfection and purification protocol for the SARS-CoV-2 S ectodomain. We describe how to trace protein yields during purification using highly sensitive and characteristic changes in S ectodomain intrinsic fluorescence upon thermal denaturation. Additionally, we detail several optimized aspects of the purification including timing and temperature. This protocol facilitates consistent, high-quality preparations of the SARS-CoV-2 S ectodomain. For complete details on the use and execution of this protocol, please refer to Stalls et al. (2022), Gobeil et al. (2022), Edwards et al. (2021), and Henderson et al. (2020).

Assessment of nucleotide/nucleoside analog intervention in primer-dependent viral RNA-dependent RNA polymerases.

Nucleotide/nucleoside analogs (NAs) are important compounds used in antiviral drug development. To understand the action mode of NA drugs, we present an enzymology protocol to initially evaluate the intervention mechanism of the NTP forms of NAs on a coronaviral RNA-dependent RNA polymerase (RdRP). We describe the preparation of SARS-CoV-2 RdRP proteins and RNA constructs, followed by a primer-dependent RdRP assay to assess NTP forms of NAs. Two representative NA drugs, sofosbuvir and remdesivir, are used for demonstration of this protocol. For complete details on the use and execution of this protocol, please refer to Wu et al. (2021).

An antibody-based proximity labeling protocol to identify biotinylated interactors of SARS-CoV-2.

Elucidating the molecular interactions between virus and host is fundamental to understanding the mechanism of viral pathogenesis. Here, we present a protocol to screen SARS-CoV-2 protein interactors using an antibody-based TurboID proximity labeling approach. This technique directly identifies biotinylated peptides labeled by the TurboID-tagged viral proteins. We describe the steps to prepare biotinylated peptide samples for mass spectrometry analysis and a stringent workflow to identify biotinylated high-confidence interactors of the virus by filtering out non-specific co-purified proteins. For complete details on the use and execution of this protocol, please refer to Zhang et al. (2022).

A computational protein design protocol for optimization of the SARS-CoV-2 receptor-binding-motif affinity for human ACE2.

The present protocol describes the computational design of the SARS-CoV-2 receptor binding motif (RBD) to identify mutations that can potentially improve binding affinity for the human ACE2 (hACE2) receptor. We focus on four positions located at the interface with the hACE2 receptor in the RBD:hACE2 complex. We conduct the design with a high-throughput computational protein design (CPD) program, Proteus, incorporating an adaptive Monte Carlo (MC) protocol that promotes the selection of sequences with good binding affinities. For complete details on the use and execution of this protocol, please refer to Polydorides and Archontis (2021).

Qualitative immunoassay for the detection of anti-SARS-COV-2 spike antibody in human milk samples.

Antibodies in milk obtained from those previously SARS-CoV-2-infected or vaccinated against COVID-19 may provide passive immunity to the breastfed infant. Few assays have been established to measure antibodies in human milk, despite the public health importance of this topic. In the present protocol, we describe an optimized indirect ELISA assay aimed to measure SARS-CoV-2-reactive antibodies in human milk, which can be used as a rapid screen on undiluted samples or to designate samples as relatively low, moderate, or high titer. For complete details on the use and execution of this protocol, please refer to Fox et al. (2020).

SARS-CoV-2 proteome microarray for COVID-19 patient sera profiling.

The immunogenicity of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) proteome is largely unknown. Here we describe a protocol for analyzing sera samples with SARS-CoV-2 proteome microarray. The proteins were expressed by either E. coli expression system or eukaryotic cell expression systems and obtained by affinity purification. The protocol includes microarray fabricating and sera profiling, which will be used to build an antibody response landscape for IgG and IgM. The protocol may help to facilitate a deeper understanding of immunity related to SARS-CoV-2. For complete details on the use and execution of this protocol, please refer to Li et al. (2021c).

Mass spectrometry and proteome analysis to identify SARS-CoV-2 protein from COVID-19 patient swab samples.

With new emerging SARS-CoV-2 strains and their increased pathogenicity, diagnosis has become more challenging. Molecular diagnosis often involves the use of nasopharyngeal swabs and subsequent real-time PCR-based tests. Although this test is the gold standard, it has several limitations; therefore, more complementary assays are required. This protocol describes how to identify SARS-CoV-2 protein from patients' nasopharyngeal swab samples. We first introduce the approach of label-free quantitative proteomics. We then detail target verification by triple quadrupole mass spectrometry (MS)-based targeted proteomics. For complete details on the use and execution of this profile, please refer to Bankar et al. (2021).

Protocol for structure determination of SARS-CoV-2 main protease at near-physiological-temperature by serial femtosecond crystallography.

The SARS-CoV-2 main protease of (Mpro) is an important target for SARS-CoV-2 related drug repurposing and development studies. Here, we describe the steps for structural characterization of SARS-CoV-2 Mpro, starting from plasmid preparation and protein purification. We detail the steps for crystallization using the sitting drop, microbatch (under oil) approach. Finally, we cover data collection and structure determination using serial femtosecond crystallography. For complete details on the use and execution of this protocol, please refer to Durdagi et al. (2021).

Facile isolation of high-affinity nanobodies from synthetic libraries using CDR-swapping mutagenesis.

The generation of high-affinity nanobodies for diverse biomedical applications typically requires immunization or affinity maturation. Here, we report a simple protocol using complementarity-determining region (CDR)-swapping mutagenesis to isolate high-affinity nanobodies from common framework libraries. This approach involves shuffling the CDRs of low-affinity variants during the sorting of yeast-displayed libraries to directly isolate high-affinity nanobodies without the need for lead isolation and optimization. We expect this approach, which we demonstrate for SARS-CoV-2 neutralizing nanobodies, will simplify the generation of high-affinity nanobodies. For complete details on the use and execution of this profile, please refer to Zupancic et al. (2021).

Domain-specific biochemical and serological characterization of SARS-CoV-2 nucleocapsid protein.

Nucleocapsid proteins are essential for SARS-CoV-2 life cycle. Here, we describe protocols to gather domain-specific insights about essential properties of nucleocapsids. These assays include dynamic light scattering to characterize oligomerization, fluorescence polarization to quantify RNA binding, hydrogen-deuterium exchange mass spectrometry to map RNA binding regions, negative-stain electron microscopy to visualize oligomeric species, interferon reporter assay to evaluate interferon signaling modulation, and a serology assay to reveal insights for improved sensitivity and specificity. These assays are broadly applicable to RNA-encapsidated nucleocapsids. For complete details on the use and execution of this protocol, please refer to Wu et al. (2021).

Identification of SARS-CoV-2 S RBD escape mutants using yeast screening and deep mutational scanning.

Here, we describe a protocol to identify escape mutants on the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spike (S) receptor-binding domain (RBD) using a yeast screen combined with deep mutational scanning. Over 90% of all potential single S RBD escape mutants can be identified for monoclonal antibodies that directly compete with angiotensin-converting enzyme 2 for binding. Six to 10 antibodies can be assessed in parallel. This approach has been shown to determine escape mutants that are consistent with more laborious SARS-CoV-2 pseudoneutralization assays. For complete details on the use and execution of this protocol, please refer to Francino-Urdaniz et al. (2021).

Fluorogenic in vitro activity assay for the main protease Mpro from SARS-CoV-2 and its adaptation to the identification of inhibitors.

This protocol describes an in vitro fluorogenic assay to measure the proteolytic activity and identify inhibitors of Mpro, the main protease produced by SARS-CoV-2 (Severe acute respiratory syndrome coronavirus 2). Studies to identify potential inhibitors of Mpro mainly rely on in silico molecular dynamics simulations or on FRET (Fluorescence Resonance Energy Transfer) substrates. The protocol is based on an aminomethyl coumarin substrate. High sensitivity, specificity, and an easily detectable fluorescent read-out are the advantages offered by this rapid assay, which allows high throughput screening of new Mpro inhibitors.