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Expression, purification, and kinetic characterization of the human strep-IDO1

Background: Tryptophan catabolism leading to T cell suppression mediated by indoleamine 2,3-dioxygenase (IDO1) is an important mechanism of tumor immune escape, and IDO1 inhibitors have attracted increasing attention as anticancer therapeutics. However, the phase III clinical trial (ECHO-301/KEYNOTE-252) of epacadostat (INCB024360) had disappointing outcomes. This revealed that purification of IDO1 with high purity is one of the major constraints that limit the development of its inhibitors.

Methods: Pan-cancer analysis was used to elucidate the relationship between IDO1 function in tumor immunology. The recombinant pET28a-IDO1-strep plasmid and E. coli Rosetta (DE3) strain were used to express and strep-tactin beads to purify the strep-IDO1 protein. High performance liquid chromatography (HPLC) was used to detect enzymatic activity of IDO1. Ten female C57BL/6 mice was used to prepared polyclonal antibody. Enzyme linked immunosorbent assay (ELISA), Western blot, and immunofluorescence were used to measure polyclonal antibody.

Results: We described an improved method for the purification of recombinant IDO1 protein based on the strep-tag using an E. coli expression system. We obtained large amount of IDO1 with enhanced purity by employing one-step purification through strep-tactin beads. The polyclonal antibody acquired immunized mice could specifically recognize both recombinant and endogenous IDO1.

Conclusions: Purified human strep-IDO1 using the protocol described in our study could be used for further biochemical and structural analyses, which may facilitate functional research and further drug screening study on IDO1.

Related Products

Cat.No. Product Name Information
S7910 Epacadostat (INCB024360) Epacadostat (INCB024360) is a potent and selective indoleamine 2,3-dioxygenase (IDO1) inhibitor with IC50 of 10 nM and displays high selectivity over other related enzymes such as IDO2 or tryptophan 2,3-dioxygenase (TDO).

Related Targets

IDO/TDO