Ferritin, a ubiquitous protein complex crucial for iron storage and metabolism, consists of Heavy (H) and Light (L) subunits with varying ratios. Mammalian Ferritin is a 24-subunit assembly with differing ratios of H and L subunits across different cellular contexts. Despite extensive study, precise assembly mechanisms and physiological functions of Ferritin remain elusive. Our research aims to investigate the H-L subunit association in Ferritin’s 24-mer structure formation. Using engineered plasmids expressing FTH fused with a SUMO tag and tag-free FTL, we prepared a 1:1 H to L ferritin heteropolymer sample. Characterization via Ni-NTA and Size Exclusion Chromatography, and SDS- and Native-PAGE confirmed self-assembly of SUMO-H-L ferritin. Findings reveal SUMO-H subunits’ association with L subunits to form a 24-mer ferritin akin to homopolymers. Cryo-EM analysis is planned to understand better how the SUMO-H and L subunits co-localize and whether there is any preferential association between H-L heterodimers versus homodimers.
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