Expression, purification and biophysical characterization of a protein fragment spanning the mature extracellular and transmembrana domains of IA2

MARÍA E. PRIMO, VALERIA A. RISSO, LAURA N.F. SOSA, MARTÍN E. NOGUERA, EDGARDO POSKUS AND MARIO R. ERMÁCORA

Buzios, Río de Janeiro, Brasil

Congreso; VII Iberoamerican Congress of Biophysics; 2009

Iberoamerican Biophysics Society

IA-2 is a protein-tyrosine phosphatase receptor located in the secretory granules of neuroendocrine cells involved in the regulation of the secretory process. IA‑2 is post‑translationally processed by proteolytic cleavage, yielding a mature extracellular domain (meIA‑2, residues 449-576), attached to the naturally inactive intracellular domain through a single transmembrane region (residues 576‑600). In a previous work we reported the X‑ray structure of two dimerizacion forms of meIA‑2 and characterized its free radical mediated oxidative degradation to yield meIA‑2 comprising residues 469‑557. Objective. The aim of the present work was to prepare, purify and biophysically characterize the me‑tmIA‑2 tandem (residues 449-600) in a folded state. Methods. His‑tag me‑tmIA‑2 was expressed in E. coli and purified by metal affinity chromatography in buffers containing 0.1% Thesit. meIA2 was prepared according to our published protocol (Primo et al. 2008, JBC). Far- and near‑UV spectroscopy measurements were carried out at 20ºC and at pH 7.0. Crosslinking was performed with DSS. Limited proteolysis was carried out with trypsin (100:1, w/w, protein/protease ratio) at 28°C, with or without 2 M urea. Results. The prepared proteins were >95% pure. His‑tag me‑tmIA2 bound to detergent micelles exhibited near and far UV-CD spectra consistent with a native fold, as judged by comparative analysis of meIA2 under identical conditions. Both proteins were similarly resistant to trypsin. Cross-linking experiments evidenced that both protein dimerize in solution. Conclusion. A protocol for the production of folded me‑tmIA2 was devised and successfully tested. me‑tmIA2 exhibits spectral features similar to those of meIA-2. These findings open the way for higher resolution studies aimed to establish the disposition and insertion mode of the extracellular domain of IA2 in artificial and natural membranes. Acknowledgments. UBA, CONICET, UNQ and ANPCyT