I have a cell line that stably expresses an EGFP-labeled GPCR. I know that most GPCRs don‘t require a signal peptide, but my protein levels are rather low, and I would like to boost translation and membrane integration of the GPCR. Do you have any recommendation?
A protein that is normally secreted in the liver is the alpha 1-antitrypsin. When there's a mutation that makes such protein aggregate in the ER, the protein is not secreted, remains in the liver and with time causes liver failure. Deficiency of such protein (because it's not secreted) causes unopposed trypsin to degrade elastin fibers in the lung causing emphysema. The mutation that causes aggregation is outside of the signal peptide. Therefore you could use alpha 1-antitrypsin signal peptide and it should work. Such facts are citable as this biology is widely known.
I agree that GPCR should not need a signal peptide; I would resort to other means to boost expression first, such as optimization of your codons, and add a WPRE to stablize your mRNA. Also, I assume you are not attaching GFP to the N-terminus of the construct, as that may cause trouble for ribosomes to direct your products to ER.
Yes, I did attach the GFP to the N-terminus of the GPCR, however, as the first transmembrane domain of the receptor serves as signal anchor sequence that directs the protein to the ER membrane, this should not be a problem per se. It might decrease the efficiency of the whole translation process a bit, which is also the reason for my question. Regarding the WPRE: mRNA stability is not the problem, the protein translation needs a little boost.
- Great discussion but I would still be very careful about manipulating N-terminus of your GOI to minimize any sorting errors. 5-10% of GPCRs do carry signal peptide but I assume you are sure yours is not one of them. If GFP has to be at the N-terminus, then as you suggested you may add a signal peptide that worked before with GFP. Here is one paper that involves secreted GFP you may want to check out: PMID: 21792177
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