Expression and characterization of SARS-CoV-2 spike proteins

Abstract

The severe acute respiratory syndrome coronavirus 2 spike protein is a critical component of coronavirus disease 2019 vaccines and diagnostics and is also a therapeutic target. However, the spike protein is difficult to produce recombinantly because it is a large trimeric class I fusion membrane protein that is metastable and heavily glycosylated. We recently developed a prefusion-stabilized spike variant, termed HexaPro for six stabilizing proline substitutions, that can be expressed with a yield of >30 mg/L in ExpiCHO cells. This protocol describes an optimized workflow for expressing and biophysically characterizing rationally engineered spike proteins in Freestyle 293 and ExpiCHO cell lines. Although we focus on HexaPro, this protocol has been used to purify over a hundred different spike variants in our laboratories. We also provide guidance on expression quality control, long-term storage, and uses in enzyme-linked immunosorbent assays. The entire protocol, from transfection to biophysical characterization, can be completed in 7 d by researchers with basic tissue cell culture and protein purification expertise.

Lower SARS-CoV-2–specific humoral immunity in people living with HIV-1 recovered from nonhospitalized COVID-19

“…The ADCP assay was modeled after prior work ( 26 , 33 ) with modifications. Briefly, quantification of ADCP was performed by covalently binding 6P spike (HexaPro) ( 39 ) to NeutrAvidin fluorescent beads (Thermo Fisher Scientific) and forming immune complexes by incubation with 1:50 diluted serum. This dilution was chosen from a 6-place 5-fold titration series starting from 1:10.…”

“…This dilution was chosen from a 6-place 5-fold titration series starting from 1:10. HexaPro was used based on its more highly stabilized trimer conformation than 2P spike ( 39 ). Monoclonal antibodies CV23 IgG1 and CV30 IgG1 ( 40 ) and CR3022 IgG1 served as positive controls, while CH65 IgG1 served as a negative control ( 41 ).…”

Nasal Nanovaccines for SARS-CoV-2 to Address COVID-19

“…The more the virus replicates, the more mutant strains are produced at the same time. In mutated virus strains, changes in the spike protein cause a greater risk of vaccine ineffectiveness [ 116 , 117 ]. Due to its strong immunogenicity, most researchers have chosen the spike protein as the research target when studying the prevention and treatment of COVID-19.…”

Intranasal SARS-CoV-2 spike-based immunisation adjuvanted with polyethyleneimine elicits mucosal and systemic humoral responses in mice

“…The dominant neutralising antibody target is the S receptor binding domain (RBD) ( Dejnirattisai et al, 2021 ; Lan et al, 2020 ). Both S and the RBD have been prepared as soluble recombinant forms which can be readily expressed and purified ( Kleanthous et al, 2021 ; Liu et al, 2020 ; Schaub et al, 2021 ), and have previously been shown to be safe and immunogenic in animal models and clinical trials ( Li et al, 2021 ; Thanh Le et al, 2020 ; Wørzner et al, 2021 ). There are currently several SARS-CoV-2-S trimer-based vaccines in development for systemic administration that use adjuvants licensed for clinical use ( Thanh Le et al, 2020 ).…”