NUSBLAT Alejandro David
congresos y reuniones científicas
EXPRESSION OF A 100kDa RECOMBINANT PROTEIN FROM Histoplasma capsulatum IN THE METHYLOTROPHIC YEAST Pichia pastoris
AGUSTINA TOSCANINI; MARIA LUJAN CUESTAS; A. D. NUSBLAT
Ciudad de Buenos Aires
Congreso; 18th International Congress on Infectious Diseases; 2018
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 Hc100, a specific protein of 100kDa from 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 Hc100 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, Westernblot and mass spectrometry.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 Westernblot using anti-histidine antibodies and by 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 encouragingthe national production of novel fungal antigens for the potential development of new rapid diagnostic tests for this clinical relevant form of the histoplasmosis disease.Keywords: Disseminated histoplasmosis, Pichia pastoris, recombinant protein, diagnoses.