@article{b25eda34802b4385afba277fcc533828,
title = "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.",
keywords = "Animals, CHO Cells, Cricetinae, Cricetulus, Gene Expression Regulation, Viral/physiology, HEK293 Cells, Humans, Models, Molecular, Protein Conformation, SARS-CoV-2, Spike Glycoprotein, Coronavirus/genetics",
author = "Schaub, {Jeffrey M.} and Chou, {Chia Wei} and Kuo, {Hung Che} and Kamyab Javanmardi and Hsieh, {Ching Lin} and Jory Goldsmith and DiVenere, {Andrea M.} and Le, {Kevin C.} and Daniel Wrapp and Byrne, {Patrick O.} and Hjorth, {Christy K.} and Johnson, {Nicole V.} and John Ludes-Meyers and Nguyen, {Annalee W.} and Nianshuang Wang and Lavinder, {Jason J.} and Ippolito, {Gregory C.} and Maynard, {Jennifer A.} and McLellan, {Jason S.} and Finkelstein, {Ilya J.}",
note = "Funding Information: We thank members of the Maynard, Finkelstein and McLellan Laboratories for providing helpful comments on the manuscript. This work was supported by NIH grant R01-AI127521 (J.S.M.), GM120554 and GM124141 (I.J.F.); the Bill & Melinda Gates Foundation INV-017592 (J.A.M., I.J.F. and J.S.M.); Welch Foundation grants F-1767 (J.A.M.) and F-1808 (I.J.F.); and the NSF 1453358 to (I.J.F.). I.J.F. is a CPRIT Scholar in Cancer Research. This research has been funded in part with federal funds under a contract from the National Institute of Allergy and Infectious Diseases, National Institutes of Health, contract number 75N93019C00050 (G.C.I.). This research was, in part, supported by the National Cancer Institute{\textquoteright}s National Cryo-EM Facility at the Frederick National Laboratory for Cancer Research under contract HSSN261200800001E. The Sauer Structural Biology Laboratory is supported by the University of Texas College of Natural Sciences and by award RR160023 from the Cancer Prevention and Research Institute of Texas (CPRIT). Publisher Copyright: {\textcopyright} 2021, The Author(s), under exclusive licence to Springer Nature Limited.",
year = "2021",
month = nov,
doi = "10.1038/s41596-021-00623-0",
language = "English (US)",
volume = "16",
pages = "5339--5356",
journal = "Nature Protocols",
issn = "1754-2189",
publisher = "Nature Publishing Group",
number = "11",
}