Over-expression and characterization of recombinant Heat Shock Protein 90 from Arabidopsis thaliana

CORIGLIANO M, MARTIN V, GOLDMAN A, LAGUIA BECHER M, CLEMENTE M

Tucumán

Conferencia; XLV Reunión Anual de la Sociedad Argentina de Investigación en Bioquímica y Biología Molecular; 2009

<!-- /* Font Definitions */ @font-face {font-family:Calibri; mso-font-alt:"Century Gothic"; mso-font-charset:0; mso-generic-font-family:swiss; mso-font-pitch:variable; mso-font-signature:-1610611985 1073750139 0 0 159 0;} /* Style Definitions */ p.MsoNormal, li.MsoNormal, div.MsoNormal {mso-style-parent:""; margin-top:0cm; margin-right:0cm; margin-bottom:10.0pt; margin-left:0cm; line-height:115%; mso-pagination:widow-orphan; font-size:11.0pt; font-family:Calibri; mso-fareast-font-family:Calibri; mso-bidi-font-family:"Times New Roman"; mso-ansi-language:ES-AR; mso-fareast-language:EN-US;} @page Section1 {size:612.0pt 792.0pt; margin:70.85pt 3.0cm 70.85pt 3.0cm; mso-header-margin:35.4pt; mso-footer-margin:35.4pt; mso-paper-source:0;} div.Section1 {page:Section1;} -->  The molecular chaperone Heat Shock Protein 90 (HSP90) plays an important role in folding, stabilization and activation processes of client proteins. For the activation of its subtrates, HSP90 binds and hydrolyzes ATP. To gain insight on this role in Hsp90 from plants, we have characterized citosolic Hsp90 isoforms from A. thaliana. AtHSP81.1, AtHSP81.2 and AtHSP81.3 codifying sequences were amplified by PCR from RAFL09-47-C2, RAFL09-06.O8 and RAFL06-10-K09, respectively and cloned in pGEM-T vector. The sequences were confirmed by sequencing and then were directionally subcloned in pRSET-A vector and expressed in E. coli Rosetta strain. In particular, recombinant AtHSP81.2 (rAtHSP81.2) over-expression 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 Vmax and 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 positive controls.