EXPRESSION OF A RECOMBINANT PROTEIN FROM Histoplasma capsulatum IN THE METHYLOTROPHIC YEAST Pichia pastoris

AGUSTINA TOSCANINI; A. D. NUSBLAT; MARIA LUJAN CUESTAS

Ciudad de Buenos Aires

Congreso; REUNIÓN CONJUNTA DE SOCIEDADES DE BIOCIENCIAS; 2017

SAIB

Histoplasmosis is a systemic and endemic mycosis widely distributedin the Americas caused by the dimorphic fungus Histoplasmacapsulatum. The infection is usually asymptomatic in immunocompetentindividuals. However, immunocompromised patients maycontract the disseminated form of the disease, which has a badprognosis and requires rapid diagnosis and treatment. The definitivediagnosis involves the isolation of H. capsulatum by culture fromclinical specimens, which may take up to 4 weeks. In addition, molecularmethods are expensive and have low sensitivity and immunoassayspresent many false-positive results. Thus, the aim of thiswork is to express a specific protein form H. capsulatum to developa novel direct immunoassay and to perform characterization studiesof this protein as a first approach for the potential development ofnovel therapeutic strategies.The gene that codes for this protein was constructed with a secretorysignal and a polyhistidine-tag and was expressed in Pichiapastoris X-33 strain. Cell culture supernatants and lysates from differentinduction times were analyzed by SDS-PAGE, Western blotand mass spectrometry. The expression was also scaled-up to 1Lusing a stirred tank bioreactor as a proof of concept for the industrialproduction.A band of the expected size was observed in the supernatants at24, 48 and 72h of methanol induction in Coomasie blue stained gelsand its identity was confirmed by Western blot using anti-histidineantibodies and mass spectrometry. The highest expression levelwas observed at 24h of induction. Also, a lower molecular weightband was observed at 48 and 72h of induction, probably due to degradationprocesses.In conclusion, P. pastoris proved to be a valid biotechnological toolfor the expression of this specific protein, thus encouraging the nationalproduction of novel fungal antigens for the potential developmentof new rapid diagnostic tests for this clinical relevant form ofthe histoplasmosis disease.Keywords: Disseminated histoplasmosis, Pichia pastoris, recombinantprotein, diagnoses, scaling-up