XI International Meeting on Paracoccidioidomycosis

XI International Meeting on Paracoccidioidomycosis

Resume:116-1

Poster (Painel)

116-1

Heterologous expression and purification of the recombinant Ras GTPase of Paracoccidioides brasiliensis

Authors:

Rodrigo Silva (UNIFESP/DIADEMA - Universidade Federal de São Paulo) ; Ana Eliza Coronel Janú Haniu (UNIFESP - Universidade Federal de São Paulo) ; Hugo Pequeno Monteiro (UNIFESP - Universidade Federal de São Paulo) ; Rosana Puccia (UNIFESP - Universidade Federal de São Paulo) ; Wagner Luiz Batista (UNIFESP/DIADEMA - Universidade Federal de São PauloUNIFESP - Universidade Federal de São Paulo)

Abstract

Paracoccidioides brasiliensis is a thermodimorphic fungus responsible for paracoccdioidiomycosis (PCM), a prevalent systemic mycosis in South America. A number of signaling pathways are associated with fungal adaptation to the host´s environment. Ras proteins are key elements in intracellular signaling and are involved in a variety of vital processes. This proteins are molecular switches that are active when GTP-bound and inactive when GDP-bound. Both processes are regulated by enzymatic reactions. In P. brasiliensis, two genes homologues of Ras (Ras1 and Ras2) have been previously characterized. In order to obtain Ras1 and Ras2 recombinant molecules, we cloned, expressed and purified the proteins. The complete coding cDNA of Ras1 (644-bp) and Ras2 (717-bp) were obtained by RT-PCR with primers added of the restriction enzymes sites, cloned and excised from pJET vector with EcoRI/HindIII and subcloned in the same sites of pET28a in frame with the sequence encoding a His6 tag. Correct in-frame ligation was checked by endonuclease restriction and sequencing. Selected plasmids were inserted into E. coli BL21 for IPTG-induced protein expression. After expression and Western blot analysis, we observed two bands with molecular weight of 28 kDa and 30 kDa, corresponding to Ras1 and Ras2, respectively. To purify the recombinant Ras1 and Ras2, individual bacterial clones were cultivated overnight at 37°C under shaking in LB medium containing kanamycin. Bacterial cell pellets were obtained by centrifugation and disrupted by sonication. Precipitates were pelleted by centrifugation and suspended in buffer containing 8 M urea. Recombinant proteins were purified by chromatography in Ni-NTA columns according to the manufacturer's instructions. In this work we obtained recombinant Ras1 and Ras2 of P. brasiliensis. It makes possible the production of the polyclonal antibody for use in immunological analysis, cytolocalization and active assays. Financial support: CNPq

Keyword: pET28a, Ras GTPase, signaling pathways

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