Dear all,
Why is a protein, which is expressed in HEK293 cells, not excreted to the medium? The protein remains soluble into the cell. Which could be the reasons?
Thanks for your time!
Best wishes,
Kika
Proteins that need to be secreted into the media by a mammalian cell should posses a signal peptide, that takes the protein through the ER by cotranslational translocation and the secretory export pathway. Proteins like interleukin-2, CD5, the Immunoglobulin Kappa light chain, trypsinogen, serum albumin, and prolactin follow this route by virtue of their signal sequences. If you need your recombinant protein to get secreted into the media, you need to add the signal sequence; usually at the amino-terminus of a protein.
You can find more information in these papers :
Article Improving mammalian cell factories: The selection of signal ...
Article Optimized signal peptides for the development of high expres...
Thanks a lot for your answer! The protein has its natural signal peptide. Should I use another signal peptide?
Hi Praveesh,
Could another cause be causing this issue or is it the signal peptide? Could I run any experiment in the lab to confirm it? Thanks a lot for your help.
Hi
Dear
It may be secreted but its concentration in the cell culture medium is too low, for detection. You can perform a fast and easy dot blot for detection of it.
thanks
Dear Enriqueta,
Be sure to know the synthetic and secretory features of your protein. Usually, a molecular address label on proteins as part of their post-translational modification is essential for their trafficking. In addition, the literature is replete, read articles on methodology as this may avail you the strategies that have been used by other researchers and you may see the need to optimize conditions.
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