ATPase ACTIVITY AND IMMUNOSTIMULATORY PROPERTIES OF Arabidopsis thaliana HSP90 EXPRESSED IN E.coli

CORIGLIANO, MARIANA G; MARTIN VALENTINA; GOLDMAN ALEJANDRA; LAGUÍA BECHER MELINA; CLEMENTE MARINA

San Miguel de Tucumán, Tucumán.

Congreso; 45th Annual Meeting. Argentine Society for Biochemistry and Molecular Biology Research; 2009

Sociedad Argentina de Investigación en Bioquímica y Biología Molecular

The molecular chaperone Heat Shock Protein 90 (Hsp90) plays an important role in folding stabilization, activation of client proteins and also mammalian and pathogen Hsp90 showed immunostimulatory properties. For substrate activation, Hsp90 binds and hydrolyzes ATP. To gain insight on the biochemical and immunological properties of plant Hsp90, we expressed citosolic Hsp90 isoforms from A. thaliana. AtHSP81.1, AtHSP81.2 and AtHSP81.3 were directionally subcloned in E. coli Rosetta (D3) strain. In particular, recombinant AtHsp81.2 (rAtHsp81.2) overexpression was confirmed by SDS-PAGE and western blot using anti-His antibodies. rAtHsp81.2 was purified by Ni-NTA affinity chromatography and its molecular weight was determined by MALDI-TOF-MS. The purification procedure yielded 2mg/ml of 99% pure protein, suitable for further structure–function studies. rAtHsp81.2-ATPase activity was measured in vitro and the V and max Km were determined. Finally, we evaluated the capacity of rAtHsp81.2 to induce in vitro proliferation of splenocytes from naive BALB/c mice. We observed that rAtHsp81.2 is able to stimulate proliferation of spleen cells as it was observed with Hsp83 of Leishamania infantum. LPS and ConA with or without polimixine B were used as controls