Ferritin is a highly conserved supramolecular nanostructure and a key player in iron homeostasis. Eukaryotic ferritins are predominantly heteropolymeric species consisting of 24 structurally similar, but functionally different subunit types named H (for Heavy, ~ 21 kDa) and L (for Light, ~ 19 kDa). These subunits co-assemble in various H to L ratios to form shell-like protein structures (isoferritins) of ~ 8 nm inner cavity diameter able of accommodating thousands of iron atoms in the form of biologically available hydrous ferric oxide mineral core. Despite their discovery more than 8 decades ago, the isoferritin nanostructure-assembly process has not been studied owing to the lack of a good expression system that could produce these nano-assemblies. Earlier in-vitro reconstitution attempts using chemical denaturation and unfolding of recombinant homopolymers H- and L-subunits followed by their renaturation have either largely failed or yielded very low amounts of heteropolymer ferritins that are not representative of those occurring naturally. Here, we developed a novel expression system that allows the synthesis of recombinant human heteropolymer ferritin of any desired H to L composition using the Gibson Assembly cloning technique. The purified heteropolymer ferritins exhibited a single band on native gel confirming protein assembly and a single protein nanostructure, and their H to L subunit composition was confirmed by SDS gel and capillary electrophoresis.

Expression and purification of human heteropolymer ferritin using a novel expression system

ACS Fall 2021

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