Perhaps the circular vector plus insert is so coiled and supercoiled that the enzymes involved in translation cannot find their binding sites resulting in lower activity than the linear molecule

Linearized plasmids may expose promoter regions more effectively, facilitating transcription machinery access and boosting gene expression.

Hi Anton,

Previous responses are valid. I'm adding a third one. I'm assuming you are measuring RNA levels via qRT-PCR. One possible issue is the presence of contaminating DNA (plasmid) co-purified with your RNA.

These will be amplified if not completely removed by a DNAse treatment step. Amplicons from linear plasmids are notoriously more easily amplified than circular ones. The extremely high expression levels you get with the linear fragment transfection (and not with the circular ones) are indeed odd.

I would expect some differences but not a black and white result (e.g. no detectable expression in transient and stable transfection).

Regardless the mechanism or technical artifact however, with those expression levels you should be able to detect vp80 in a lysate of transfected cells simply by Coomassie staining.

If true, your linearised fragment is producing similar expression levels to a full blown infection, which is remarkable but also suspicious.

Hope it helps

Francesco