Hey, I have a pcDNA 3.1 plasmid with a murine trypsinogen 7 sequence. I tried various transfection methods (lipofectamine2000, FuGene, Electroporation) into HEK 293T cells, but I had very low relative expression (qPCR: only 50 time higher than negativ control) and really low amount of protein. Therefore I can´t detect thetrypsinogen on my western blots.
My transfection control (gfp) shows a good efficiency.
What can I optimize to increase my protein expression?
I am also trying to express/purify a recombinant protein using HEK293FT which is a fast growing version of HEK293 and have struggled through many obstacles.
Following are lessons I learned.
1. Using a codon optimized gene results in higher expression than non-codon optimized.
2. My recombinant protein has secretion signal which allows secretion of expressed proteins, thus no need for cell disruption and dealing with intracellular proteins for purification.
3. I use 25 kDa linear PEI as transfection agent which is really cheap compared to any lipid based transfection agents, and the transfcetion is comparable.
4. I use Freestyle 293 expression medium, which is serum free and helps in purification process fro the culture supernatant.
5. Just as the cell numbers are important (about 60-70% confluence), the amount of media is also important. For example I use to use 50 ml medium in 75cm2 flask, which turned out to be suffocating my cells. Apparently cells need oxygen and CO2 more than they need the medium. So I only use 20-25 ml medium when growing cells in 75cm2 flask.
6. Supernatant should be harvested around day 4 post transfection to maximize protein expression and minimize cell death and release of intracellular proteases.
7. Valproic acid: Use of valproic acid at 4mM concentration significantly increases expression of proteins.
https://www.ncbi.nlm.nih.gov/pubmed/18454496
Good luck,
Hi Franziska G Thiel ,
from what you said it seems more likely a promoter issue, I suppose that your GFP (transfection control) is under the same promoter you are using for your trypsinogen (if not just change promoter), but maybe it is mutated into the promoter sequence during your plasmid preparation (if it come from a single bacterial colony it can happen to pick the unlucky one).
The options suggested in the previous answers seem to me very good but it doesn't seem to me a problem of transfection actually, it's more a problem of expression. If there's nothing wrong with your plasmid I agree it could be a problem of codon usage and if you are still not convinced on the transfection efficiency just order new primers designed on the backbone and compare the plasmid quantity present in a DNA extraction from your GFP vs trypsinogen transfected cells.
Good luck!
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