Avaliação de estratégias de produção em cultivo descontínuo da proteína quimérica multi-antigênicaTgAGS/BsT de toxoplasma gondii em escherichia coli BL21 star

Abstract

Toxoplasmosis, despite advances in science and technology, is a disease that requires attention since there is no vaccine capable of immunizing humans and animals against all isolated types of Toxoplasma gondii. Thus, the use of chimeric proteins, which can contain multiple antigens, is a very promising alternative for the process of obtaining a vaccine and diagnostic test for toxoplasmosis due to the great diversity of antigens presented by T. gondii. In this context, the main focus of the present study is to evaluate batch culture strategies in the production of the multi-antigenic chimeric protein TgAGS/BsT from Toxoplasma gondii. To achieve the proposed objective, several exploratory cultures were initially carried out to observe the kinetic behavior of E. coli BL21 Star in five different medium compositions (2xTY, TB supplemented with glucose, TB supplemented with glycerol, LB and M9) without the addition of IPTG (inductor). Then, cultures of E. coli B21 Star were carried out with 1.0 mM IPTG at different times of initiation of induction (0.5, 1 and 6 h) to evaluate the effects on cell growth, production of the protein of interest, pH and acetic acid formation. The results showed that among the culture media evaluated, 2xTY and TB supplemented with glycerol had the best cell concentration values of 3.42 ± 0.05 g/L and 5.48 ± 0.05 g/L, respectively. In the assays induced by IPTG, a higher expression of TgAGS/BsT protein was observed, with induction beginning within 6 h of culture, with a maximum concentration of protein of interest of 1.82 ± 0.02 g/L for the 2xTY and 2 medium. 2.49 ± 0.03 g/L for the TB medium. In addition, later induction by IPTG provided greater stability of plasmid pET-TgAGS, remaining with values above 90% at the end of culture. The expression of the TgAGS/BsT protein was confirmed by electrophoresis, with a molecular mass of approximately 18.6 kDa. Therefore, the present study identified the best conditions of culture medium composition and induction time for the production of TgAGS/BsT protein in rotary incubator cultures, with results that encourage the expansion to cultures in larger capacity bioreactors.