Expression of a recombinant protein from histoplasma capsulatum in the methylotrophic yeast Pichia pastoris

TOSCANINI MA; NUSBLAT AD; CUESTAS ML

Congreso; Reunión conjunta de Sociedades de Biociencias; 2017

Histoplasmosis is a systemic and endemic mycosis widely distributed in the Americas caused by the dimorphic fungus Histoplasma capsulatum. The infection is usually asymptomatic in immunocompetent individuals. However, immunocompromised patients may contract the disseminated form of the disease, which has a bad prognosis and requires rapid diagnosis and treatment. The definitive diagnosis involves the isolation of H. capsulatum by culture from clinical specimens, which may take up to 4 weeks. In addition, molecular methods are expensive and have low sensitivity and immunoassays present many false-positive results. Thus, the aim of this work is to express a specific protein form H. capsulatum to develop a novel direct immunoassay and to perform characterization studies of this protein as a first approach for the potential development of novel therapeutic strategies.The gene that codes for this protein was constructed with a secretory signal and a polyhistidine-tag and was expressed in Pichia pastoris X-33 strain. Cell culture supernatants and lysates from different induction times were analyzed by SDS-PAGE, Western blot and mass spectrometry. The expression was also scaled-up to 1L using a stirred tank bioreactor as a proof of concept for the industrial production. A band of the expected size was observed in the supernatants at 24, 48 and 72h of methanol induction in Coomasie blue stained gels and its identity was confirmed by Western blot using anti-histidine antibodies and mass spectrometry. The highest expression level was observed at 24h of induction. Also, a lower molecular weight band was observed at 48 and 72h of induction, probably due to degradation processes. In conclusion, P. pastoris proved to be a valid biotechnological tool for the expression of this specific protein, thus encouraging the national production of novel fungal antigens for the potential development of new rapid diagnostic tests for this clinical relevant form of the histoplasmosis disease.