An alarm system for biomedical construct design: a lesson from the unintended protein product of eGFP

Hidden open reading frames in plasmids are present in about 6% of constructs, predominantly in the +2 frame, with some producing stable proteins like eGFP.

• Why it matters: Unintended protein products from hidden ORFs can affect gene expression and safety, especially in sensitive applications like gene therapy, yet they are poorly characterized.
• What they did: A computational pipeline was developed and applied to analyze 6,308 eGFP-containing plasmids, revealing hidden ORFs in approximately 6% of cases, mainly in the +2 frame.
• The result: The findings enable better detection of unintended proteins, helping to prevent unexpected outcomes in biomedical constructs and improve safety in gene-based therapies.