Heterologous Expression and Immunoreactivity of Wheat Amylase–Trypsin Inhibitors

Wheat amylase–trypsin inhibitors (ATIs) are known for their allergenic properties, but their precise role in celiac disease and other wheat-related disorders remains unclear. Pure ATI preparations provide a basis for detailed biochemical and immunologic characterization; however, the complexity of isolating ATIs in the absence of other immunoreactive wheat proteins is an ongoing challenge. To address this limitation, bacterial and yeast heterologous expression systems were evaluated for the production of pure ATI preparations. While the synthesis of recombinant ATIs 0.28 and CM3 in Escherichia coli was challenging regarding protein solubility, promising results were observed for ATIs 0.19, CM1 and CM3 expressed in Pichia pastoris, both in terms of solubility and protein yields. The biochemical and immunoreactive properties of these recombinant ATIs were evaluated by SDS-PAGE, MALDI-TOF MS, LC-MS/MS, circular dichroism analysis, and in vitro inhibitory and immunoassays, and compared to native ATIs isolated from wheat. Our findings shed light on the difficulties in isolating pure ATIs and provide valuable insights into the potential impact of these proteins on human health. By using innovative expression systems, we aim to contribute to the detailed biochemical and immunogenic characterization of ATIs and improve the understanding of their role in wheat-related disorders and associated allergic responses.