The Cys rich extracellular fragment of IA2: expression in E. coli and preliminary characterization

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

Buzios, Río de Janeiro, Brasil

Congreso; VII Iberoamerican Congress of Biophysics; 2009

Iberoamerican Biophysiscs Society

IA-2 is tyrosine phosphatase receptor that targets to the insulin‑secreting granules of pancreatic b cells. This protein was first identified as a major autoantigen in insulin-dependent diabetes mellitus. Since then, it has been found involved in several crucial steps of insulin production and secretion. It is a large and complex protein with several intra and extracellular domains. Among these are a PTPase domain that faces the cytoplasm, a single‑pass transmenbrane domain, a mature extracellular domain that faces the granule interior, and an N‑terminal cysteine‑rich fragment (ntcrIA2) that is proteolytically removed as the granule mature and which ulterior fate is unknown. The structure of the PTPase domain was determined earlier. The X‑ray structure of the mature extracellular domain was recently solved in our laboratory. The structure of processed N‑terminal fragment remains a mystery and no sequence similarity to any structurally characterized protein could be inferred from it. The aim of this work was to produce ntcrIA2 as a recombinant protein to initiate its chemical and biophysical characterization. The entire IA-2 gene was used as a template to amplify a cDNA encoding amino acids 35 to 131. The product was cloned into pET-9 and used to transform Bl21(DE3)RIL-CodonPlus cells. Transformed cells were cultured and protein expression induced. The over expressed recombinant protein was recovered as inclusion bodies from cell lysates, purified with high yield under denaturing conditions, and refolded by dilution to a physiological buffer. The identity of the produced protein was confirmed by ES‑MS. ntcrIA2 contains four cysteine residues, which are deemed to form disulfide bridges in the native state. The refolding condition to form the appropriate disulfides and its chemical characterization are underway. The successful production of ntcrIA2 provides an unique opportunity to study its structure and get insights on its biological function.