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.

Amberlite has been shown to be an appropriate material for the adsorption of organic contaminants from aqueous solutions. In addition, Amberlite XAD-2 has been successfully used, as an alternative to Bio-Beads, to remove Triton X-100 from protein solutions, such as from samples of solubilized membrane proteins. However, Amberlite has not been tested as an adsorbent when a mixture of detergents is necessary to solubilize and refold a target protein. Here the authors show that Amberlite XAD-4 can be appropriately used to aid the purification process of proteins solubilized from inclusion bodies with the ternary detergent system consisting of Sarkosyl, Triton X-100 and CHAPS.