Cloning, production, purification and characterisation of recombinant peptide-protein fusions

Cysteines-rich macrocyclic peptides show great promise as therapeutics, but their recombinant production faces significant biochemical challenges. To address this, we evaluated cysteine-free proteins as fusion partners for cysteines-rich macrocyclic peptides to enhance both solubility and expression levels in Pichia pastoris. Initially, a (hACE2)-targeting macrocyclic peptide model was used to evaluate candidate cysteine-free tags as alternatives to HaloTag, which contains an unpaired cysteine residue. The analysis identified a promising tag that yielded high expression levels and robust solubility for both N- and C-terminal fusions. Cell-binding assays confirmed that the hACE2-targeting macrocyclic peptide retained high binding affinity for hACE2-expressing cells, with binding affinity values comparable to HaloTag's. This strategy was later applied to a macrocyclic peptide targeting CD19, a membrane protein target in B- cell malignancies. These peptides were successfully produced and purified in Pichia pastoris. A self-competition assay validated their functionality, demonstrating that pre-incubated anti-CD19 peptides inhibited CD19 binding to yeast cells displaying the same peptide, confirming specific epitope binding and correct topology. Further studies are ongoing to assess their functionality on target cancer cells. In conclusion, this study identifies and validates an effective cysteine-free fusion protein for the recombinant expression of macrocyclic peptides in P. Pastoris.