Hello Mr Lee,

You can do "overexpression" transfection.

When construct the plasmid use one of the housekeeping gene's promoter instead of your target protein's promoter.

Thanks, Okan, for your suggestion. I thought about it, but changing the promoter will be my last solution. I am thinking of co-tranfecting the HeLa and H1299 cells with a T-antigen expressing plasmid or adding the proteasome inhibitor MG132 a few hours before harvesting the cells. I am still looking for people's input.

Dear Clement, have you already though about switching to mRNA?

https://viromer-transfection.com/mrna-transfection

Best,

Sandra

Thank you very much, Sandra. It is a good idea.

If I were you, I would first test expression of your target protein in the cell lysate (by blotting for your protein). If you have poor expression then definitely consider changing promoter, based on my poor experience, I can say EF promoter should give you much higher expression than CMV at least in HELA and HEK cells (higher the expression rate more secretion?) Or

If you see good expression in the cells, you might have secretion issue, then think about your secretion signal and target protein size are compatible or not.

Good luck.

Thank you very much, Tuvshintugs.

I found a paper on comparison of different promoters:

https://doi.org/10.1371/journal.pone.0010611