The main text details the experimental design, including the generation of CO and CPO ADAMTS13 variants using different algorithms, and the evaluation of these variants in terms of protein expression, structure, function, immunogenicity, and cellular bioenergetics. The study employed cell-free in vitro translation, cellular expression experiments, circular dichroism (CD) for structure determination, Seahorse respiration assays for bioenergetics, and MHC-associated peptide proteomics (MAPPs) for immunogenicity assessment.
Key findings from the study include:

1. Protein Expression and Stability: CO and CPO variants showed differences in translation kinetics and solubility compared to WT. CO had higher extracellular expression but was less stable in cells, while CPO had lower extracellular expression but maintained similar stability to WT.
2. Enzymatic Activity and Substrate Binding: CO variants exhibited altered enzymatic kinetics, with higher Vmax and Km values, indicating lower affinity to VWF compared to WT and CPO. CPO, on the other hand, had similar binding affinity and kinetics to WT.
3. Protein Structure and Folding: CD analysis predicted similar secondary structure composition but revealed differences in folding dynamics, suggesting potential differences in protein stabilities.
4. Cellular Bioenergetics and ER Stress: CO and CPO-expressing cells showed higher ATP production and maximal oxygen consumption rates compared to WT. CO variants induced higher ER stress, as evidenced by increased levels of ER stress markers BiP and phosphorylated-eIF2α.
5. Immunogenicity and Glycosylation: Recoded ADAMTS13 variants presented distinct MHC-II peptides and elicited different CD4+ T-cell proliferation responses. Glycosylation profiles also differed, with CO and CPO variants showing more complex glycans and altered glycosylation sites compared to WT.

The study highlights the multifaceted impact of synonymous gene recoding on protein properties, including expression, stability, structure, function, immunogenicity, and cellular bioenergetics. While recoding strategies can enhance protein expression, they may also introduce undesirable changes that affect protein safety and efficacy. The findings underscore the importance of thorough evaluation of recoded transgenes within the context of host cell characteristics to optimize the design of protein therapeutics and mitigate potential risks associated with gene recoding. The work entitled “ Therapeutic implications of synonymous gene recoding: insights into mechanisms controlling protein biogenesis and activity” was published on Protein & Cell (published on 2025).
DOI：https://doi.org/10.1093/procel/pwaf028