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

Authors

Katarzyna I Jankowska, Upendra K Katneni, Brian C Lin, Hannah M Brenner, Randilu Amarasinghe, Nigam Padhiar, Patrick Dopler, Nobuko Hamasaki-Katagiri, Wells W Wu, Haojie Zhu, Hideki Taguchi, Je-Nie Phue, Paul Rahul, Sean Smith, Luis Santana-Quintero, Ofer Kimchi, Ryan C Hunt, Nicholas C Hurst, Emilee Knowlton, Nikolai F Schwabe, Anton A Komar, Jeremy W Fry, Chava Kimchi-Sarfaty

Published in

Trends in biotechnology. Oct 01, 2026. Epub Oct 01, 2026.

Abstract

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.

PMID:
42823207
Bibliographic data and abstract were imported from PubMed on 02 Oct 2026.

Read full publication at:
Please sign in to see all details.

Stats

1-terrible, 9-excellent. How would you rate this publication? Sign in in to submit your rating.

Post a comment

You need to be signed in to post comments. You can sign in here.

Comments

There are no comments yet.