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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 Statement N.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).

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