High-yield expression of properly folded insulinome-associated protein intracellular domain (IA-2ic) in E. coli.

MAURICIO P. SICA,; MARÍA E. PRIMO; MARIO R. ERMÁCORA; EDGARDO POSKUS

BIOTECHNOLOGY AND APPLIED BIOCHEMISTRY

Ed. Portland Press Ltd

Lugar: Gran Bretaña; Año: 2003 vol. 37 p. 301 - 309

0885-4513

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 immuno-reactivity 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.