I overexpressed a particular protein in CHO cells and I'ld like to know how to detect/know/confirm that secretion is the bottleneck for it's low yiled/production ?
One point from my side is:
Measure the intracellular and extracellular protein concentration. If intracellular protein concentration is more than extracellular protein concentration then i can confirm that secretion is the bottleneck for low yield/production.
I'ld like to know what all the other methods to detect.
Compare intracellular protein vs. secreted protein over time. If secretion is the bottleneck, the intracellular amount should increase, if synthesis is the limit, the intracellular amount should be in a steady state after an induction period. Might be somewhat difficult, though, if you can't induce the expression but it is always on. Maybe nutrient starvation can do the trick.
To stop secretion, you may cool down your cells to below 4°C.
You also could try immunostaining of your cells to detect the whereabouts of your protein.
Dear Vikram
you can try to compare secretion vs intracellular protein production but you can do it only if you have a specific antibody able to recognize the intracellular protein, because while the extracellular could be detected by SDS-page since the extracellular media do not contain many other proteins, it is not so easy with the intracellular.
I think is more productive spend effort to try to optimize the yield of secreted fraction that try to undestand how much prrotein is inside, because it peovide you an information but not a solution.
In general, when i observe low yields in the surnatant, i try to:
- replace the signal peptide:
- redesign the protein domain.
- change expression conditions (eg 32°C instead 37°C)
- order a codon optimized gene (in case that my sequence present a codon bias problem)
good luck
Manuele
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