Expression, purification and in silico characterization of a 100kda protein from Histoplasma capsulatum

NUSBLAT AD; NOSEDA DG; CUESTAS ML; TOSCANINI MA

Congreso; 15TH INFOCUS; 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. Objectives: The aim of this work is to express Hc100, a specific protein of 100kDa 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.Methods: The gene that encodes for Hc100 was constructed with a secretory signal and a polyhistidine-tag and was expressed in the methylotrophic yeast 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 using a 6 L stirred-tank bioreactor as a proof of concept for the industrial production. Purification of Hc100 from a 24 h cell culture supernatant was carried out using a Ni-NTA affinity chromatography column and flow-throws and eluates were analyzed by SDS-PAGE and Western blot.Results: A band of the expected size was observed in the supernatants at 24, 48 and 72 h 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 24 h of induction. Also, a lower molecular weight band was observed at 48 and 72 h of induction, probably due to degradation processes. Hc100 from a 24 h cell culture supernatant was purified with a 90% of purity. Conclusions: 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.