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

SICA M. P., PRIMO M. E., ERMÁCORA M. R., POSKUS E.

BIOTECHNOLOGY AND APPLIED BIOCHEMISTRY

Portland Press Ltd

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 glutatione 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 wich proved to be highly specific and sensitive.