I use suspension HEK293 [GnT1-] cells for large scale (>5L) expression of mammalian membrane proteins. My goal is to consistently have "crystallization yields" of protein. Right now I'm quite happy with my yield (~1mg/L of crude fusion protein, 0.1-0.3mg/L of final purified protein - don't laugh, we're talking about eukaryotic membrane proteins here!!) But my transient transfection protocol is quite laborious (and costly).
I'm using 25kD linear PEI to form in situ DNA:PEI complexes (based on work from the Wurm group, see link). The problem is that this method requires a very high density of cells (~20 x 10^6 cells/ml), which becomes a huge pain when dealing with sterile concentration of multiple liters of cells (via centrifugation and resuspension). Additionally, the excessive amounts of expensive media seems unnecessarily wasteful.
Instead of the high-density technique, I am getting increasingly interested in using preformed PEI-DNA complexes, thus obviating the need to excessively concentrate the cells prior to transfection. The problem is that, in my hands, expression is >10-fold lower using this method, despite varying the incubation time, PEI:DNA ratio, pH, etc etc...
Any advice for increasing my transfection efficiency using the preformed-complex technique? What is your secret sauce???
http://onlinelibrary.wiley.com/doi/10.1002/bit.21596/abstract
Sorry for meddling but is there any reason why you don't want to use a stable transfection protocol? Since you need a high cell number if you use a stable transfected cell line you would only have to grow the cells and never transfect again. If you are interested I can tell you about a system that I used for non adherent cells that worked amaizingly for me. I needed recombinant mammal expressed protein for biological assays.
@Andre: Thanks for the reply. Certainly I would like to generate stable cell lines eventually. However, I have multiple isoforms to work with, all of which I am still making cloning alterations to (mutagenesis, truncation of termini & putative unstructured regions, etc). Once I have a construct in which I am more confident then I will take the plunge into stable cell lines, since as I understand it takes quite some time & labor to create stable lines, right?
That being said, I'd be interested in the technique that you said worked well - I'm trying to absorb as much knowledge as possible about mammalian expression. Thanks again
Dear Justin
The system that I used and showed great results is a tetracycline induced system called Flp-In™ T-REx™ System (Thermo Fisher Scientific). They have different cell lines with a Flp Recombination Target (FRT) site into the genome of the mammalian cell line of choice. This saves you time and effort of generating the cell line yourself. Basically you just insert your gene of interest into pCDNA5 FRT/TO, then you transfect the desired cell with this vector and the gene is inserted into the specific FRT site. You only need to select positive transformants with antibiotic pressure but you don't need to select clones because all cells will show the same integration site. After selection you can make a cell stock and use it as a normal cell line. Among the different cell lines they have a Jurkat cell that allows suspension cell expression. Take a look at the link I'm attaching.
https://www.thermofisher.com/br/en/home/references/protocols/proteins-expression-isolation-and-analysis/protein-expression-protocol/generation-of-isogenic-expression-cell-lines-using-flp-in-t-rex-system.html
Hi Justin,
I use pre-formed PEI-DNA complexes to transfect adherent HEK293 cells. Something that we've noticed is that below a certainty cell density (~50% confluence of an adherent culture), the PEI is toxic to the cells and results in quite a lot of cell death and very unhappy-looking cells! Above that cell density, there doesn't seem to be a toxic effect. We also see the same effect with COS-7 cells, with a slightly different threshold.
We've never really been able to explain why this happens, but I wonder if there's a similar effect going on in your system. If you're trying to do the transfections at lower cell density, you might be getting less expression due to cellular stress/death.
Do you visually inspect your suspension cells, measure cell viability or otherwise monitor their "health" in some way?
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