Abstract
The COVID-19 pandemic has led to accelerated efforts to develop therapeutics and vaccines. A key target of these efforts is the spike (S) protein, which is metastable and difficult to produce recombinantly. Here, we characterized 100 structure-guided spike designs and identified 26 individual substitutions that increased protein yields and stability. Testing combinations of beneficial substitutions resulted in the identification of HexaPro, a variant with six beneficial proline substitutions exhibiting ~10-fold higher expression than its parental construct and the ability to withstand heat stress, storage at room temperature, and three freeze-thaw cycles. 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.
Highlights
Prefusion stabilization tends to increase the recombinant expression of viral fusion glycoproteins, possibly by preventing triggering or misfolding that results from a tendency to adopt the more stable postfusion structure
To generate a prefusion-stabilized SARS-CoV-2 spike protein that expresses at higher levels and is more stable than our original S-2P construct [18] we analyzed the SARSCoV-2 S-2P cryo-EM structure (PDB ID: 6VSB) and designed substitutions based upon knowledge of class I viral fusion protein function and general protein stability principles
One common strategy to stabilize class I fusion proteins is to covalently link a region that undergoes a conformational change to a region that does not via a disulfide bond
Summary
Prefusion stabilization tends to increase the recombinant expression of viral fusion glycoproteins, possibly by preventing triggering or misfolding that results from a tendency to adopt the more stable postfusion structure. To generate a prefusion-stabilized SARS-CoV-2 spike protein that expresses at higher levels and is more stable than our original S-2P construct [18] we analyzed the SARSCoV-2 S-2P cryo-EM structure (PDB ID: 6VSB) and designed substitutions based upon knowledge of class I viral fusion protein function and general protein stability principles. Substitutions of each category were identified that increased expression while maintaining the prefusion conformation (Fig. 1 and 2A).
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