Recombinant expression and purification of Plasmodium heme detoxification protein in E. coli: Challenges and discoveries.

Heme, a toxic by-product of Plasmodium's proteolytic digestion of host hemoglobin, is detoxified by the malaria parasite through its conversion into hemozoin (Hz)-the malaria pigment. This detoxification pathway is a key target for many antimalarial drugs, which aim to induce heme-mediated toxicity to the parasite. The Heme Detoxification Protein (HDP) plays a central role in heme-to-Hz transformation; however, its precise mechanism remains unclear, largely due to the absence of successful recombinant expression in a native, soluble form. In this study, we aimed to express HDP recombinantly in its native soluble state using an E. coli-based system. A range of strategies were employed, including expression of orthologs, consensus sequence design, fusion to solubility-enhancing partners, co-expression with molecular chaperones, and extensive construct optimization through N-terminal truncations. Despite extensive efforts, most recombinant HDP constructs were either insoluble or formed soluble aggregates. Notably, only a single construct-with a 44-residue N-terminal truncation and a C-terminal 6-His tag (HDPpf-C10)-was successfully expressed in a soluble form. Surprisingly, HDPpf-C10, although retaining domains implicated in heme binding and transformation, exhibited no detectable heme-to-Hz transformation activity. This finding highlights the essential role of the flexible-unstructured N-terminal region in mediating both heme binding and its subsequent conversion to Hz, providing new insights into HDP function and guiding future structural and mechanistic studies.