Journal Article

Expression and characterization of SARS-CoV-2 spike proteins

Schaub, Jeffrey M., DiVenere, Andrea M., Javanmardi, Kamyab, Hjorth, Christy K., Maynard, Jennifer A., Goldsmith, Jory, Ippolito, Gregory C., Nguyen, Annalee W., Wang, Nianshuang, Finkelstein, Ilya J., Wrapp, Daniel, Byrne, Patrick O., Lavinder, Jason J., Johnson, Nicole V., McLellan, Jason S., Chou, Chia-Wei, Kuo, Hung-Che, Hsieh, Ching-Lin, Ludes-Meyers, John, Le, Kevin C.

2021

Overview

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. The authors present a protocol for expressing and biophysically characterizing rationally engineered SARS-CoV-2 spike proteins in Freestyle 293 and ExpiCHO cell lines.

• Publisher: Nature Publishing Group UK,Nature Publishing Group
• Subject: 631/250/590/2294 / 631/45/612/1256 / Analytical Chemistry / Animals / Biological Techniques / Biomedical and Life Sciences / Cell culture / Cell lines / CHO Cells / Computational Biology/Bioinformatics / Coronaviruses / COVID-19 / Cricetinae / Cricetulus / Critical components / Evaluation / Fusion protein / Gene Expression Regulation, Viral - physiology / Health aspects / HEK293 Cells / Humans / Immunoassays / Life Sciences / Medical protocols / Membrane fusion / Membrane proteins / Microarrays / Models, Molecular / Organic Chemistry / Proline / Protein Conformation / Protein purification / Proteins / Protocol / Quality control / SARS-CoV-2 / Severe acute respiratory syndrome coronavirus 2 / Spike Glycoprotein, Coronavirus - genetics / Spike Glycoprotein, Coronavirus - metabolism / Spike protein / Structure / Therapeutic targets / Tissue culture / Transfection / Viral diseases / Viral proteins / Workflow