Structure-Based Design with Tag-Based Purification and In-Process Biotinylation Enable Streamlined Development of SARS-CoV-2 Spike Molecular Probes

Abstract

Biotin-labeled molecular probes, comprising specific regions of the SARS-CoV-2 spike, would be helpful in the isolation and characterization of antibodies targeting this recently emerged pathogen. To develop such probes, we designed constructs incorporating an N-terminal purification tag, a site-specific protease-cleavage site, the probe region of interest, and a Cterminal sequence targeted by biotin ligase. Probe regions included full-length spike ectodomain as well as various subregions, and we also designed mutants to eliminate recognition of the ACE2 receptor. Yields of biotin-labeled probes from transient transfection ranged from ~0.5 mg/L for the complete ectodomain to >5 mg/L for several subregions. Probes were characterized for antigenicity and ACE2 recognition, and the structure of the spike ectodomain probe was determined by cryo-electron microscopy. We also characterized antibody-binding specificities and cell-sorting capabilities of the biotinylated probes. Altogether, structure-based design coupled to efficient purification and biotinylation processes can thus enable streamlined development of SARS-CoV-2 spike-ectodomain probes.

Deep Mutational Scanning of SARS-CoV-2 Receptor Binding Domain Reveals Constraints on Folding and ACE2 Binding

“…The SARS-CoV-1 RBD contains a third glycan, but its introduction at the homologous N370 in SARS-CoV-2 is mildly deleterious for expression ( Figure S5 F). However, there are other surface positions where introduction of NxS/T glycosylation motifs is tolerated or even beneficial for RBD expression ( Figures S5 G–S5I); adding glycans at some of these sites could be useful in resurfacing RBDs as antibody probes ( Wu et al., 2010 ; Zhou et al., 2020c ) or epitope-focused immunogens ( Duan et al., 2018 ; Eggink et al., 2014 ; Jardine et al., 2016 ; Kulp et al., 2017 ; Weidenbacher and Kim, 2019 ).…”

Cryo-EM Structures Delineate a pH-Dependent Switch that Mediates Endosomal Positioning of SARS-CoV-2 Spike Receptor-Binding Domains

“…The protein was then applied to a Superdex 200 column in PBS, after which the spike containing fractions were pooled and concentrated to 1 mg/ml. Single chain Fc tagged RBD and NTD domains were expressed in the same manner, and purified using capture by Protein A resin, followed by cleavage of the tag using HRV3C 16 and gel filtration.…”

“…To provide insight into the stability of the spike over the course of virus-cell entry, we used differential scanning calorimetry (DSC) to measure the unfolding enthalpy of the soluble trimeric ectodomain (spike), which included GSAS and PP mutations and the T4 phage fibritin trimerization domain 4,16 , as a function of pH. Notably, at physiological pH we observed spike to be at a minimum of unfolding enthalpy (73.3 kcal/mol) or ~10% the normalized unfolding enthalpy of the average globular protein 17 ( Fig.…”

A platform incorporating trimeric antigens into self-assembling nanoparticles reveals SARS-CoV-2-spike nanoparticles to elicit substantially higher neutralizing responses than spike alone

“…5a-c, Supplementary Table S1). For this construct, we used the prefusion stabilized version of spike developed by McLellan and colleagues 24 , which included GSAS and PP mutations and the T4 phage fibritin trimerization domain along with a single-chain Fc tag for purification as described by Zhou and colleagues 38 . The conjugation mixture was loaded onto an SEC column to purify the conjugated nanoparticle product LuS-N71-SpyLinked-CoV-2 spike from unconjugated LuS-N71-SpyTag and SARS-CoV-2 spike-SpyCatcher (Fig.…”