A wheat Germin-like protein expressed in Escherichia coli has antifungal activity against Fusarium solani

MARCHETTI, M.F.; MANSILLA, A.Y.; CONDE, R.D.; MENDIETA, J.R.

Mar del Plata

Congreso; X Congreso Argentino de Microbiología General SAMIGE; 2014

A Germin-like Protein with trypsin inhibitor activity has been purified from intercellular fluid of wheatleaves and it was named Germin-like Protein Inhibitor (GLPI). Since GLPI is a heat-resistant protein, aheating step (30 min at 70ºC) is included in the protocol of purification. In addition to its inhibitoractivity, GLPI has at least two more enzimatic activities: superoxide dismutase (SOD) and adenosineglucose pyrophosphatase/phosphodiesterase (AGPPase). Moreover, its antifungal activity againstplant fungal pathogens was confirmed recently. Also, the expression of GLPI (rGLPI) in E. coli wasachieved. 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 thenew protocol was called rGLPI70. To confirm the homogeneity of the eluted fraction and to confirm itsidentity, the purified protein was analyzed by SDS-PAGE and protein gel blotting followed byinmunodetection with antiserum against GLPI isolated from wheat. The purified protein showed oneclear band of the expected molecular mass (21 kDa). With the objective to study whether rGLPI andrGLPI70 maintain the antifungal activity observed with the native protein, we analyzed the effect ofboth on F. solani spores germination. In vitro quantitative assays showed that 240 ug/ml of rGLPI andrGLPI70 inhibited a 70 % of F. solani spores germination. These results were similar to thoseobtained for native GLPI. In conclusion, the modified protocol consists in an easier, faster and lessastringent alternative to obtained rGLPI. The rGLPI antifungal activity against F. solani suggests thatthis protein could be used to develop new natural pesticides.