Synthetic Intrinsically Disordered Proteins Enable Soluble Expression of Disulfide-Rich Therapeutics in <i>E. coli</i>

We previously reported the de novo design of three small (<20 kDa), highly soluble synthetic intrinsically disordered proteins (SynIDPs) and demonstrated their utility as solubility tags for proteins and antifouling agents. Building on this work, we now show that these hypersoluble SynIDPs significantly enhance the soluble expression of disulfide-rich proteins (DRPs) of therapeutic relevance, including fibroblast growth factor 21 (FGF-21), interleukin-15 (IL-15), and bovine pancreatic trypsin inhibitor (BPTI). Through SynIDP fusions, we achieve soluble recombinant production of functionally active DRPs containing a single disulfide bond in the E. coli strain BL21(DE3) and up to three nonconsecutive disulfide bonds in the E. coli SHuffle T7 Express strain, without the need for refolding. The resulting SynIDP-DRP fusion proteins retain biological activity, confirming correct folding and disulfide-bond formation with minimal interference from the SynIDP tag, which obviates the need for tag removal. Collectively, these findings highlight the versatility and utility of SynIDPs as molecular tools to advance the production and development of protein therapeutics.