Expression of antimicrobial snakin-1 peptide in insect cells

ALMASIA, NATALIA INÉS; MOLINARI PAULA; NARHIRÑAK, VANESA; TABOGA OSCAR; VAZQUEZ-ROVERE, CECILIA

Congreso; XLVII Reunión Anual de la SAIB; 2011

Snakin-1 (StSN1) is a broad-spectrum antimicrobial cysteine-rich peptide isolated from Solanum tuberosum. Although it is active against fungal and bacterial pathogens, no information is available concerning the mechanism of action. Only some members of its family were expressed probably because its activity affects prokaryotic system viability. Even recombinant rSN1 could be expressed in E.coli, it was localized in inclusion bodies and displayed distinct activities than plant StSN1. In order to obtain purified protein for biochemical studies and subsequent research, we expressed SN1 in Spodoptera frugiperda (Sf9) insect cells using the Bac-to-Bac baculovirus expression system. This system allows expression of toxic proteins while the correct folding is more likely maintained because of their extensive and more accurate post-translational modifications. Here, the gene sequence codifying for the mature StSN1 peptide was subcloned into the pFastBacTMHT B, transposed into the bacmid and virus were amplified. Tagged-SN1 expression was detected in the nuclei fraction of infected Sf9 cells, recombinant protein was purified and mice were immunized to obtain polyclonal antibodies. More purifications are being carried out for biochemical experiments and for testing the protein on a wide variety of phytopathogens to assess the antimicrobial potential of rSN1 from baculovirus expression system.