PT  - JOURNAL ARTICLE
AU  - Ching-Lin Hsieh
AU  - Jory A. Goldsmith
AU  - Jeffrey M. Schaub
AU  - Andrea M. DiVenere
AU  - Hung-Che Kuo
AU  - Kamyab Javanmardi
AU  - Kevin C. Le
AU  - Daniel Wrapp
AU  - Alison Gene-Wei Lee
AU  - Yutong Liu
AU  - Chia-Wei Chou
AU  - Patrick O. Byrne
AU  - Christy K. Hjorth
AU  - Nicole V. Johnson
AU  - John Ludes-Meyers
AU  - Annalee W. Nguyen
AU  - Juyeon Park
AU  - Nianshuang Wang
AU  - Dzifa Amengor
AU  - Jennifer A. Maynard
AU  - Ilya J. Finkelstein
AU  - Jason S. McLellan
TI  - Structure-based Design of Prefusion-stabilized SARS-CoV-2 Spikes
AID  - 10.1101/2020.05.30.125484
DP  - 2020 Jan 01
TA  - bioRxiv
PG  - 2020.05.30.125484
4099  - http://biorxiv.org/content/early/2020/05/30/2020.05.30.125484.short
4100  - http://biorxiv.org/content/early/2020/05/30/2020.05.30.125484.full
AB  - The COVID-19 pandemic caused by the novel coronavirus SARS-CoV-2 has led to accelerated efforts to develop therapeutics, diagnostics, and vaccines to mitigate this public health emergency. A key target of these efforts is the spike (S) protein, a large trimeric class I fusion protein that is metastable and difficult to produce recombinantly in large quantities. Here, we designed and expressed over 100 structure-guided spike variants based upon a previously determined cryo-EM structure of the prefusion SARS-CoV-2 spike. Biochemical, biophysical and structural characterization of these variants identified numerous individual substitutions that increased protein yields and stability. The best variant, HexaPro, has six beneficial proline substitutions leading to ∼10-fold higher expression than its parental construct and is able to withstand heat stress, storage at room temperature, and multiple freeze-thaws. A 3.2 Å-resolution cryo-EM structure of HexaPro confirmed that it retains the prefusion spike conformation. High-yield production of a stabilized prefusion spike protein will accelerate the development of vaccines and serological diagnostics for SARS-CoV-2.Competing Interest StatementN.W. and J.S.M. are inventors on U.S. patent application no. 62/412,703 (Prefusion Coronavirus Spike Proteins and Their Use). D.W., N.W. and J.S.M. are inventors on U.S. patent application no. 62/972,886 (2019-nCoV Vaccine). C.-L.H., J.A.G., J.M.S., C.-W.C., A.M.D., K.J., H.-C.K., D.W., P.O.B., C.K.H., N.V.J., N.W., J.A.M., I.J.F., and J.S.M. are inventors on U.S. patent application no. 63/032,502 (Engineered Coronavirus Spike (S) Protein and Methods of Use Thereof).