Anais do Congresso Paranaense de Microbiologia
IV Congress of Microbiology in Paraná

The Coronavirus Disease 19 (COVID-19) is characterized by a severe acute respiratory syndrome of great importance. The etiological agent is the SARS-CoV-2 virus, which has 29 proteins in its structure. Among them, the Spike protein, a surface glycoprotein which is responsible for the virus entry into the host cells, through its affinity for the Angiotensin Converting Enzyme 2 (ACE2). Due to this feature, the use of S recombinant protein has shown that this protein is an important target with great applicability in studies of vaccines and diagnostic methods. Therefore, the aim of this work was to standardize the purification process of recombinant S protein expressed in Escherichia coli. Employing the sequence retrieved from the National Center for Biotechnology Information database (NCBI), predicted immunogenic epitope regions were analyzed and used to artificially synthesize a recombinant plasmid. The clone was used to the heat shock transform Escherichia coli BL21-star (DE3) competent cells. The production of the recombinant protein fused to histidine tail was induced at 37ºC in a medium supplemented with IPTG 1 mM for 4 hours. Then, the bacterial culture was centrifuged, and the pellet was lysed by sonication method with lysis buffer containing urea, due to the protein has been expressed as an insoluble protein. Afterwards, it was centrifuged again, and the supernatant was collected. The protein purification was performed through a chromatography column containing nickel salts, which has affinity with the histidine tail. To obtain the proteins, an eluent solution containing imidazole was used at different concentrations (10%, 20%, 30%, 40%, 50%, 75% and 100%). The protein analysis was performed by 13% SDS-PAGE gel and the quantification occurred by the Bradford method. After the separation of proteins eluted fractions through SDS-PAGE gel, a great amount of this proteins was observed in the eluates of 20% and 30% concentrations. The protein quantification confirmed this analysis with results of 1,7 mg/mL for 20% and 0,7 mg/mL for 30%. Therefore, it was concluded that the solution containing imidazole at a concentration of 20% has better results for the S recombinant protein purification, demonstrating the importance of standardizing the purification process of this protein, to improve the production and its use in the future application.