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Biotechnology and Applied Biochemistry (2003) 37, (301–309) (Printed in Great Britain)

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High-yield expression of properly folded insulinoma-associated protein intracellular domain (IA-2ic) in Escherichia coli

Mauricio P. Sica*, María E. Primo*, Mario R. Ermácora†‡¹ and Edgardo Poskus*†

*School of Pharmacy and Biochemistry, University of Buenos Aires and IDEHU-CONICET, Buenos Aires, Argentina, †Department of Science and Technology, National University of Quilmes, Roque Sáenz Peña 180, (1876) Bernal, Argentina, and ‡Consejo Nacional de Investigaciones Científicas y Técnicas, Argentina

Key words: autoimmunity, diabetes, ELISA, immunological marker, islet cell antigen (ICA), recombinant protein expression.

Abbreviations used: bcELISA, blank-corrected ELISA; IA-2, insulinoma-associated protein; IA-2A, IA-2-directed autoantibodies; IA-2ic, IA-2 intracellular domain; ICA512, islet cell antigen 512; RBA, radiobinding assay.

¹To whom correspondence should be addressed, at the Department of Science and Technology, National University of Quilmes (e-mail ermacora@mail.unq.edu.ar).

The intracellular domain of insulinoma-associated protein (IA-2), IA-2ic, is a prominent antigen in autoimmune diabetes, and autoantibodies to it are early markers of the disease. The high-yield expression of properly folded IA-2ic is needed for basic research and crucial for low-cost immunoassays aimed at the detection of these autoantibodies in diagnostic and preventive medicine. In previous work, the expression of IA-2ic fused to glutathione S-transferase or to a biotinylatable peptide was reported; however, these methods had very poor yield. Here we show that, utilizing a codon-optimized gene, up to 80 mg of pure and properly folded autoantigen per litre of Escherichia coli culture may be obtained. Furthermore, the addition of a C-terminal His-tag greatly facilitates IA-2ic purification without compromising either its immunoreactivity or its expression yield. To take advantage of the recombinant antigen, an enzyme immunoassay format was developed which proved to be highly specific and sensitive.

Received 7 October 2002/19 November 2002; accepted 6 January 2003

Published as Immediate Publication 6 January 2003, DOI 10.1042/BA20020090

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