High-yield production in Escherichia coli and convenient purification of a candidate vaccine against SARS-CoV-2.

OBJECTIVES: The aim of the present work was to identify a time-saving, effective, and low-cost strategy to produce in Escherichia coli a protein chimera representing a fusion anti-SARS-CoV-2 candidate vaccine, consisting of immunogenic and antigenic moieties. RESULTS: We overexpressed in E. coli BL21(DE3) a synthetic gene coding for CRM197-RBD, and the target protein was detected in inclusion bodies. CRM197-RBD was solubilized with 1 % (w/v) of the anionic detergent N-lauroylsarcosine (sarkosyl), the removal of which from the protein solution was conveniently accomplished with Amberlite XAD-4. The detergent-free CRM197-RBD was then separated from contaminating DNA using polyethylenimine (PEI), and finally purified from PEI by salting out with ammonium sulfate. Structural (CD spectrum) and functional (DNase activity) assays revealed that the CRM197-RBD chimera featured a native and active conformation. Remarkably, we determined a yield of purified CRM197-RBD equal to 23 mg per litre of culture. CONCLUSIONS: To produce CRM197-RBD, we devised the use of sarkosyl as an alternative to urea to solubilize the target protein from E. coli inclusion bodies, and the easy removal of sarkosyl by means of Amberlite XAD-4.

Production in Escherichia coli of recombinant COVID-19 spike protein fragments fused to CRM197.

During 2020, the COVID-19 pandemic affected almost 108 individuals. Quite a number of vaccines against COVID-19 were therefore developed, and a few recently received authorization for emergency use. Overall, these vaccines target specific viral proteins by antibodies whose synthesis is directly elicited or indirectly triggered by nucleic acids coding for the desired targets. Among these targets, the receptor binding domain (RBD) of COVID-19 spike protein (SP) does frequently occur in the repertoire of candidate vaccines. However, the immunogenicity of RBD per se is limited by its low molecular mass, and by a structural rearrangement of full-length SP accompanied by the detachment of RBD. Here we show that the RBD of COVID-19 SP can be conveniently produced in Escherichia coli when fused to a fragment of CRM197, a variant of diphtheria toxin currently used for a number of conjugated vaccines. In particular, we show that the CRM197-RBD chimera solubilized from inclusion bodies can be refolded and purified to a state featuring the 5 native disulphide bonds of the parental proteins, the competence in binding angiotensin-converting enzyme 2, and a satisfactory stability at room temperature. Accordingly, our observations provide compulsory information for the development of a candidate vaccine directed against COVID-19.