The Impact of Cysteine Substitutions on TGF-β3 Expression, Purification, Folding, and Activity

faces significant challenges due to TGF-β3's propensity for misfolding and aggregation, driven by a high disulfide bond content and low aqueous solubility. To address these limitations, the impacts of substituting non-conserved cysteine residues C7, C16 and C77 with serine on TGF-β3 folding, dimerization and activity were investigated. Whilst C7 and C16 form an intra-chain disulfide bond, C77 forms an inter-chain disulfide bond stabilizing dimer formation. Our results showed that the C7S, C16S double cysteine mutant protein exhibited reduced aggregation, increased dimer formation, and maintained wild-type biological activity in nano-luciferase reporter gene assay. In contrast, both C77S single and C7S, C16S, C77S triple mutants were purified predominantly in monomeric forms and displayed about 2.5-fold reduced activities. Our findings highlight the roles of the non-conserved C7, C16 and C77 cysteine residues in TGF-β3 folding and aggregation. The identification of the C7S, C16S mutant as a more soluble protein with wild-type TGF-β3 activity offers a promising strategy for improving recombinant TGF-β3 production to facilitate therapeutic applications. This study underscores the importance of targeted cysteine engineering to overcome the inherent challenges associated with the production of TGF-β3 and related complex disulfide-rich proteins.