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Development of therapeutics to this target has previously relied on native purifications of Pf20S, which is challenging and has limited the scope of previous efforts. Here, we report an effective recombinant Pf20S platform to facilitate drug discovery. Proteasome assembly was carried out in insect cells by co-expressing all fourteen subunits along with the essential chaperone homolog, Ump1. Unexpectedly, the isolated proteins consisted of both a mature and an immature complex. Cryo-EM analysis of the immature complexes revealed structural insights detailing how Ump1 and the propeptides of the \u03b22 and \u03b25 subunits coordinate \u03b2-ring assembly, which differ from human and yeast homologs. Biochemical validation confirmed that \u03b21, \u03b22, and \u03b25 subunits of the mature proteasome were catalytically active. Clinical proteasome inhibitors, bortezomib, carfilzomib and marizomib were potent but lacked Pf20S selectivity. However, the tripeptide-epoxyketone J-80 inhibited Pf20S \u03b25 with an IC\n                  <jats:sub>50<\/jats:sub>\n                  of 22.4 nM and 90-fold selectivity over human \u03b25. Structural studies using cryo-EM elucidated the basis for the selective binding of J-80. Further evaluation of novel Pf20S-selective inhibitors such as the reversible TDI-8304 and irreversible analogs, 8304-vinyl sulfone and 8304-epoxyketone, confirmed their potency and selectivity over the human constitutive proteasome. This recombinant Pf20S platform facilitates detailed biochemical and structural studies, accelerating the development of selective antimalarial therapeutics.\n                <\/jats:p>","DOI":"10.1101\/2025.08.13.670186","type":"posted-content","created":{"date-parts":[[2025,8,15]],"date-time":"2025-08-15T04:25:19Z","timestamp":1755231919000},"source":"Crossref","is-referenced-by-count":4,"title":["A recombinant expression system for the\n                  <i>Plasmodium falciparum<\/i>\n                  proteasome enables structural analysis of its assembly and the design of selective 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