Expressão do fragmento RBD da proteína S1 do vírus SARS-Cov-2 em leveduras

Abstract

The pandemics Corona Virus Disease started in 2019 (COVID-19) has being the third outbreak of coronaviruses in the last two decades. Initially associated to cases of pneumonia, the contamination was related to a seafood market in Wuhan City, China. The transmission of the virus occurs from person to person, directly or indirectly. SARS-Cov-2 belongs to the Coronaviridae family and the β-coronavirus genus. The virus genome is a single RNA strand positive of 30 kilobases (kb). The surface of the virus is composed of 3 main proteins, spike (S), membrane (M) and envelope (E). The receptor binding domain (RBD) is located in the S1 subunit. The viral RBD binds to angiotensina conversor enzyme (ACE 2) and then the virus can enter into the cells. The production of recombinant RBD becomes interesting as it can be used directly for the production of antibodies. The aim of this work is to produce the recombinant RBD of the S1 protein from SARS-Cov-2 for studies of immunological response. The yeast Pichia pastoris strains GS115 and Glycoswitch were used as expression systems, which implicates in different glycosylations of the antigen. GS115 determinates a glycosylation that can be beneficial as adjuvant, while the Glycoswitch strain, modified to reproduce humanized glycosylation, can induce a more specific immunity. The RBD protein was recovered from the supernatant and purified by metal affinity chromatography. The elution fractions were analyzed by SDS-PAGE and Western Blot. The yeast Pichia pastoris proved to be a good system to obtain the soluble recombinant RBD, but with low expression. New strategies to optimize the production are being evaluated. For example, a new strain of P. pastoris, X33 has been cloned to tests the expression and evaluate the yields.