CONICET | Buscador de Institutos y Recursos Humanos

A Germin-like Protein with trypsin inhibitor activity has been purified from intercellular fluid of wheat leaves and it was named Germin-like Protein Inhibitor (GLPI). Since GLPI is a heat-resistant protein, a heating step (30 min at 70ºC) is included in the protocol of purification. In addition to its inhibitor activity, GLPI has at least two more enzimatic activities: superoxide dismutase (SOD) and adenosine glucose pyrophosphatase/phosphodiesterase (AGPPase). Moreover, its antifungal activity against plant fungal pathogens was confirmed recently. Also, the expression of GLPI (rGLPI) in E. coli was achieved. The original protocol includes proteins resolubilization from inclusion bodies using 6M urea. Although this compound is removed by followings steps of the protocol, a modification was applied. After induction, E. coli cells were pelleted and the soluble fraction was heated at 70 ºC for 30 minutes. The heat-resistant proteins were loaded in a nickel matrix and the recombinant protein obtained by the new protocol was called rGLPI70. To confirm the homogeneity of the eluted fraction and to confirm its identity, the purified protein was analyzed by SDS-PAGE and protein gel blotting followed by inmunodetection with antiserum against GLPI isolated from wheat. The purified protein showed one clear band of the expected molecular mass (21 kDa). With the objective to study whether rGLPI and rGLPI70 maintain the antifungal activity observed with the native protein, we analyzed the effect of both on F. solani spores germination. In vitro quantitative assays showed that 240 ug/ml of rGLPI and rGLPI70 inhibited a 70 % of F. solani spores germination. These results were similar to those obtained for native GLPI. In conclusion, the modified protocol consists in an easier, faster and less astringent alternative to obtained rGLPI. The rGLPI antifungal activity against F. solani suggests that this protein could be used to develop new natural pesticides.