Obtenção do domínio proteico RBD do SARS-CoV-2 da variante Gama expresso em duas diferentes linhagens de Komagataella phaffii, em fermentador

Abstract

By the end of 2022, more than 5.3 billion doses of vaccines against COVID-19 had already been administered, however, the inequality in the distribution of these vaccines has as consequence the continuity of the virus circulation, favoring its evolution and given the origin of new variants that can evade the immunological protection conferred by the available vaccines. Therefore, it is necessary to generate alternative technologies to meet the demand for vaccines and protective therapies. Current vaccines are based on the Spike (S) protein, due to its importance in virus entry into cells and viral transmission, conferring its role as a target for antibody neutralization. The development of vaccines against the new variants focuses on the receptor binding domain (RBD) of the protein S to the host cell receptor. Recombinant protein-based vaccines are important because they can be manufactured inexpensively in large-scale production facilities using microbial hosts, such as Komagataella phaffii. This methylotrophic yeast can use methanol as a carbon source due to the presence of alcohol oxidase (AOX) enzyme genes. The strong AOX promoters are used in genetic construction for efficient expression of heterologous proteins. In order to obtain higher levels of production, it is desirable to use cultures at higher cell densities in fermenters, where it is possible to control the flow of methanol, which plays a key role in inducing the expression of the protein of interest. In this context, the main objective of our work was to obtain the RBD protein of the Gamma variant (RBDmut) of SARS-CoV-2 expressed in the GS115 and GlycoSwitch strains of Komagataella phaffii, evaluating the culture parameters that affect the metabolism of the microorganism and the expression. The tests were carried out in a 1 L fermenter with different methanol feed flows during 48 hours, the induction phase, monitoring temperature and dissolved oxygen. The RBDmut protein was recovered in the culture supernatant and purified by metal affinity chromatography, and the elution fractions were analyzed by SDS-PAGE and Western Blot. The results demonstrated a higher protein recovery in the culture submitted to a higher methanol feed flow, in a fraction drained after 26 hours of induction for both yeast strains. However, the expression of RBDmut in all cultures was lower when compared to previous cultures conducted in shaker, expressing the RBD of the original strain, Wuhan. These clones were generated using a much smaller plasmid, which suggests the insertion of a higher number of copies of the gene of the genoma of the yeast. This study contributed to point out several aspects to be considered in the generation of new recombinants of K. phaffii clones and for the determination of important parameters in the fermentation processes.