Dear everyone,
I am trying to express the extracellular domain of a membrane protein in HEK 293F cells. The construct contains the signal peptide of the protein itself. However, the protein does not exctete into the media but retains inside the cells. Do you also encounter such a situation and do you know any tricks to make the protein excrete?
With many thanks!
Dear Yeping? did you cloned only the extracellular domain and the signal peptide?
Yes. I only cloned the extracellular domain and the signal peptide. @ Manuele Martinelli
Did you check the subcellular localisation of your protein?
One possibility is that the construct does not properly fold/glycosylate and aggregates or is retained by the quality control system. Another is that your construct accidentally unmasked an alternative targeting signal, e.g. an ER retention signal.
Hi, it might help to replace the original SP with another one. Kappa chain SP or Albumin SP are very often used.
@Annemarie Honegger Thank you. Could you tell me what is the most effective way to check the subcellular localisation of your protein? Add a GFP tag to the protein and check it by a confocal microscope? Isolation cell fractions and check it by WB?
Actually, the construct, in which a motif containing multiple glycosylation sites is deleted in order to make the protein more stable, is identical to the one in a publication. However, that paper shows that the construct can secretory expression. The only difference is that the construct is expressed in CHO lec cells, but I expressed it in 293F cells. Do you think that the different expression pattern is related to the different cell lines? Do you think keeping the highly glycosylation motif would make it secrete in 293F cells?
@Rolf Fendel
Thank you. I am also considering change the SP. I heard that IL-2 SP is also often used. Would Kappa chain SP or Albumin SP be better that IL-2 SP?
Expression level can change a lot with the SP chosen. Do not forget to remove the other SP completely, two SP are mostly non-functional.
- Badges
- Science method
- Similar topics
- Biological Science
- Genetics
- Gene Expression
- Protein Expression