Synonymous sequence design controls the folding, processing, and activity of the therapeutic protein ADAMTS13.

Synonymous recoding is widely used to enhance therapeutic protein expression, yet its broader effects on biogenesis and function remain unclear. We systematically examined how synonymous recoding shapes protein structure, processing, and activity using plasma protease ADAMTS13 (a disintegrin and metalloprotease with thrombospondin type 1 repeats, member 13) and its truncated MDTCS (metalloprotease, disintegrin-like, thrombospondin type 1, cysteine-rich, and spacer) variant across cell-free expression, transient transfection, lentiviral delivery, and targeted genomic integration. Wild-type, codon-optimized, and codon-pair-optimized constructs displayed pronounced, system-dependent differences in intracellular proteolysis, secretion, enzymatic performance, and post-translational modifications. Targeted genomic integration produced the highest protein fidelity, whereas transient and lentiviral systems yielded lower extracellular protein levels, particularly for codon-pair-optimized variants. Glycoproteomic analyses revealed distinct glycosylation patterns among synonymous constructs, indicating altered maturation, while Ankyron-based assays detected differences in molecular surface properties of recoding variants. Optimized MDTCS exhibited increased catalytic activity but reduced substrate affinity. These findings demonstrate that synonymous recoding can reshape proteostasis and activity, emphasizing the need for comprehensive evaluation of engineered therapeutic genes beyond yield.