Prokaryotic expression and purification of inorganic nitrogen transporters in wheat

Abstract:

The nitrate transporter (NRT) and ammonium transporter (AMT) are crucial transmembrane proteins involved in the absorption, transport, and distribution of inorganic nitrogen in wheat. Obtaining NRT/AMT and preparing corresponding antibodies are conducive to probing into their tissue localization and comprehending the nitrogen utilization process in wheat. In this study, four genes (TaNPF4.5, TaNPF8.3, TaNRT3.1, TaAMT1.2) with high expression levels were chosen and cloned from 405 genes of the TaNRT/TaNPF family and 23 genes of the TaAMT family identified previously. The transmembrane domains of the four transporters were predicted by HMMER to determine the putative expression segments, followed by prokaryotic expression and purification. Under the induction with 1 mmol/L IPTG at 37 ℃, the non-transmembrane segments of TaNPF4.5, TaNPF8.3, and TaNRT3.1 reached the highest expression levels (as inclusion bodies) after 4 h, while TaAMT1.2 was expressed at the highest level (as a soluble protein) after 3 h. TaNPF4.5, TaNPF8.3, and TaNRT3.1 were purified by a pH gradient. The purity of TaNPF4.5 and TaNPF8.3 reached about 87% and 85% at pH 2.0 and pH 3.0, respectively, both of which were suitable for antibody preparation. However, the purity of TaNRT3.1 did not reach 85%. TaAMT1.2 was purified by an imidazole gradient, reaching the purity of about 95% at 20 mmol/L imidazole, and the antibody was prepared successfully. The expression, purification, and antibody preparation of TaAMT1.2 not only provides insights into the expression, purification, and antibody preparation of membrane proteins including TaNPF4.5 and TaNPF8.3 but also lays a foundation for studying the expression and localization of membrane proteins in wheat.