I'm trying to produce membrane protein using HEK293S cells.
I have tested both transient expression with PEI and stable cell line and found transient expression can allow us to obtain higher amount of target protein.
Now I want to try large scale expression with transient transfection of HEK293S cells in Suspension with PEI.
We have 500 ml flask and 1 L spinner flask.
Is there anyone who know the appropriate method?
Initial transient expression was performed with adherent cells.
I have read several papers about my questions, but nothing mentioned about large scale method.
So I need your suggestions.
check this video :
http://www.jove.com/video/51897/recombinant-protein-expression-for-structural-biology-hek-293f
Recombinant Protein Expression for Structural Biology in HEK 293F Suspension Cells: A Novel and Accessible Approach
Nicola Portolano1, Peter J. Watson1, Louise Fairall1, Christopher J. Millard1, Charles P. Milano1, Yun Song1, Shaun M. Cowley1, John W.R. Schwabe1
I produced a fragment of the LDL receptor by transient transfection of HEK 293 GNT-/- cells with PEI.
In my experience there are some important parameters to optimize:
-The volumen of culture should be no more than 1/4 of the flask volume.
-We obtained better results with 70 % humidity than with 90-95 %. We used 5 % CO2 and 150 rpm as culture conditions.
-No antibiotics should be present on the transfection.
-The cell culture medium is extremly important. The Free Style 293 Expression Medium works much better than any other medium we tested (unfortunately, is very expensive).
-The PEI/DNA ratio should be optimized. In our case 3:1 worked well. We worked with hybridoma medium for transfection.
-The cell density that works better with your protein is also very variable (we tested 1e6 to 20e6 cells/ml iwhen performing the transfection, obtaining better results when using 4e6).
-We harvested the cell after 4 days expression.
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