Stable vs transient - which gives the highest yield? - I'm setting up a expression system for a secreted protein, should I choose s (Nov/13/2006 )
I want to express a soluble protein in a hek cell line. It needs to be hek because I want to be sure the glycosylation is right.
Now, I don't know if I should count on transient or stable transfection. I need huge amounts, 50-100 mg of pure protein. I have been looking through the literature and found that specific transient expression systems can give about 20 or more mg/L of medium. I found one paper about a stable cell line giving 5 mg/L. The "problem" with the transient is that a complicated vector construct i used, which involves parts from 3 different vectors. Furthermore, as far as I can deduce from various papers, it doesn't seem that episomal amplification vector/cell systems are much better than non episomal replicating ones, am I right on that?
I'd think about a constitutive strong promoter in vector more than a transient or stable transfection. What promoter does your plasmid has? Now, thinking on if the transfection is stable, it could be that your plasmid inserts in a region of silenced DNA, so maybe transient transfection could be better. Anyway, I think you must try both types of transfection.
you need to consider reproducibility and cost too.
A stable cell line will approx express continous level of prot.
So for preparing exp and for reproducing it,that's good
Second one : efficent transfection is not cheap... but culturing more cells is cheaper