Dear Shuaichen,

From what you have said, the mature protein that your cells are producing is (as far as you can tell) identical to the wild-type protein. The only issue is whether the change to the N-terminus, before the signal peptide, will affect the processing. My gut feeling is that it will not; however, the processing of proteins in the ER/Golgi is complex, and it will be a lot of effort to rule out the possibility of a subtle alteration, for example to glycosylation. 

If I were you, I would use the cell line that you already have for working up your downstream experiments without delay. Mostly likely the peptides that you produce will be fine. In the meantime, I would prepare a new construct without the FLAG tag (site directed mutagenesis should allow you to do this quickly), and make a new cell line with this. When you have the new cell line, you can use that for the "publication" experiments, based on the work up with the other cell line. Otherwise, I would fear that you will have a problem on publication with an easy target for referees; and you will never really be sure that your protein is OK. Hopefully this would give you a good balance of making progress in your research and feeling confident that your data are sound.

Hope this helps, even if it's not what you wanted to hear!

Hi Nicholas,

Thanks for your reply! I'm making the new construct with the Flag at C terminal. I saw people adding Flag at C terminal is safer but also not 100% guarantee.

Hi Shuaichen,

Expressing the flag epitope at the amino-terminus in front of the signal peptide of your secreted protein will block it from being translated at the rough ER - it will be cytoplasmic instead, and not proteolytically processed nor secreted. You should still be able to detect it with the M2 antibody by IF of the expressing culture or in a Western using whole cell lysate.

No guarantees of protein function with the epitope tag at the C terminus, but it should be processed and secreted properly!

Hi Craig,

Thank you so much for your answer! Can you give me a reference that says exactly express the meaning

"Expressing the flag epitope at the amino-terminus in front of the signal peptide of your secreted protein will block it from being translated at the rough ER - it will be cytoplasmic instead, and not proteolytically processed nor secreted."

Just want to cite this reference and when others ask me the same question I can show them. Thanks!

Hi Shuaichen,

Attached is both a review and a link to the appropriate section in Lodish et al MoBio of the Cell (NCBI textbook). Not much detail in either I am afraid; maybe a RGer has some better suggestions.

Basicly, signal peptides (SP) can be broken into Type I and II. I is located at the very N-terminal and are typically 18-22 residues but may range from 5-50 residues. Type II are less common and are internal in the peptide and often occur in combination with Type I SPs for transmembrane proteins rather than secreted proteins; they are prominent in channels and receptors with multiple transmembrane domains.

When the first few residues of a Type I signal peptide pops out of the ribosome, a docking complex recognizes and binds it, pauses translation, and transports it to the Rough Endoplasmic Reticulum, where translation is again activated, pushing the SP and the rest of protein through the membrane into the lumen. The surveillance for Type I SPs is only for the N-terminus. Adding something before the SP turns it into a regular cytoplasmic translated protein.

Article Protein Secretion and the Endoplasmic Reticulum

Hi Craig,

Thank you so much for your reply! I use a website to check the SP position, the prediction is the first 23 amino acid is the SP..

You explain quite clear how the whole secretion process is. Can I understand like this, for the stable cell I made (a Flag added at N terminus of a secreted protein), this protein cannot be secreted. It will stay in the cytoplasma with the Flag tag and because of the lack of Golgi complex proceeding, this protein may not be mature. In other words, I may not detect the protein with its own antibody.

Besides lentivirus vector, I also have the same sequence in a pCDNA3 vector, I did a transient transfection to overexpress the protein in 293T. My result is I can detect the correct protein size with anti-Flag but cannot detect with its own antibody, can I assume this is because of the N terminus Flag that inhibit the protein maturation?

Thanks!

Hi Shuaichen,

The insertion of the Flag epitope in front of the N-terminal signal peptide will prevent it from being secreted. It will be cytoplasmic. I am not completely sure what you mean by 'mature' as it includes many aspects of protein synthesis. It will not be secreted, the signal peptide will not be cleaved off, post-translational modifications that occur in the ER and Golgi will not occur (glycosylation, phosphorylation, disulphide isomerization, etc). BUT... most antibodies don't care about that type of stuff since they only recognize a small epitope (typically ~8 aa residues), and so you may still be able to detect the protein in a whole cell lysate but not in the medium since it will not be secreted.

Lentivirus or pCDNA3, does not matter. I use unpackaged, transfected lentivirus vector to test for expression of proteins from new constructs in 293T cells - all rules still apply for transient expression as long as the DNA gets into the cell. Packaging lentivirus vector just makes it easier to get into hard to transfect cells. But I am not clear about what you mean by 'can detect the correct protein size with anti-Flag' - is this in cell lysate or in the medium (secreted protein)? And methodology? Western or Immunofluorescence? Is your 'own antibody' certified for the technique that you are using? Many antibodies are limited in their specific uses.