Recombinant indoleamine 2,3-dioxygenase as a reliable alternative to HRP with enhanced cumulative output in...

Horseradish peroxidase (HRP) is the most widely used reporter in immunoassays, yet its recombinant production and the heterogeneity of isoforms derived from plants impose constraints and practical inconveniences on its application. Here, we report the development of human indoleamine 2,3-dioxygenase (IDO) as a reliable, heme-containing recombinant peroxidase alternative. The recombinant IDO was efficiently expressed in Escherichia coli. While the capture-step purification yielded over 60 mg/L of protein with >90% purity, further purification provided monomeric IDO with >98% purity. Functional assessments revealed that IDO exhibits enhanced signal persistence compared to HRP. At 48 s, the IDO system maintained a residual signal 35-fold higher than HRP, resulting in a comparable total photon output over the observation period. Kinetic analysis demonstrated that although IDO has lower catalytic efficiency, it can be effectively compensated by increasing enzyme loading to achieve a maximal reaction rate (V_max) comparable to HRP. Furthermore, Western blot with IDO-antibody conjugates showed stable bands intensities during extended exposures (5 min), matching the performance of conventional HRP. This sustained signal output offers a distinct advantage for applications requiring prolonged acquisition, such as high-throughput screening and the detection of low-abundance proteins. Overall, these results demonstrate that human IDO is a feasible and cost-effective recombinant product that can serve as a potential substitute for the traditional plant-derived HRP.