I´m working with a chimeric receptor that I´m trying to express in HEK cells, I´ve designed three constructs and I clone them into the pGEM4Z-A120-EGFP vector, for two of my constructs I have detected the expression on the cells, but for one of them I can not detect anything not by flow cytometry neither by WB.
I´m trying to fuse the extracellular domain from one receptor with the intracellular domain of another one, but one of theme is a type 1 membrane protein and the other one is a type 2.
For the construct that it didn't work I was thinking that maybe could need a signal peptide in order to be translated correctly, but at this point I don't know if that is a possibility, do you think that it could be viable to add this signal peptide to the construct that i already have or is not that easy?
If I understand correctly, you could try this: clone in a small piece of DNA which codes for the signal peptide into your vector. You would have to get the DNA seq synthesised (and the reverse comp. strand - and including the appropriate restriction sites and an ATG etc if you want it at the N -term) and get it HPLC purified, then simply anneal the two strands using a PCR machine (ie heat at 92 deg. for 30 sec, anneal at 65 deg or close to the m.p. for 5 mins, cool to 4 deg, Tris pH 8.5 buffer will do). Then digest and ligate as you would normally. I have done this with a 100+bp sequence successfully, to get the tag I wanted...
I already had a plasmid with the appropriate signal sequence, and was able to use to following method to add that signal sequence to an another clone:
Synthesize a long primer which contains (from 5' to 3') the 2nd strand of the 5' end of your receptor and the 2nd strand of the 3' end of your signal sequence. 20 or so bases from each sequence should suffice. Using the signal sequence clone as template, PCR using primers directed against the 5' end of the signal sequence (upstream primer) and the long primer (downstream primer). This product will thus contain your signal sequence plus the 5' end of your receptor. Purify the PCR product, then mix this template with your receptor template and PCR using primers against the 5' end of the signal sequence (upstream primer) and the 3' end of your receptor (downstream primer). Clone by method of choice.
^^ what they said, but remember that the signal sequence you add doesn't have to be the one that appears on the protein naturally. You may wish to try several, to accommodate the machinery of the cell you're expressing in. We've used the human IL-2 leader successfully, and the human IFNL3 one also.
I'd agree that overlap PCR is the easiest way to add a leader.
Good Luck!
F
Thank you very much to all (Richard, Robert and Grant), I think the overlapping PCR is a good approach for my experiments, so I will try that. You were very helpful.
I will let you know how everything goes or whether if I have more doubts.
I wish you all a nice day.
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