GPCR expression in HEK293Unable to get :(
Posted 19 July 2013 - 02:33 AM
Does anyone have the similar experience with expressing GPCRs in cell lines? Is the problem because these are the transmembrane proteins? I remember reading in some papers that “receptor transport protein” is required for surface expression of some olfactory receptors in HEK293 cells. Has anyone followed any such strategy for successful expression of transmembrane protein?
Thanks a lot
Posted 24 July 2013 - 09:40 AM
Posted 05 August 2013 - 10:49 PM
HEK-293 cells are used routinely in our lab for expression of mammalian GPCRs. Could the lack of expression be related to your target protein not being a mammalian protein? Or, is it the "N1" vector construct. Certain mammalian GPCRs require the tag on the "C"-terminus. Some proteins express better with the tag on the "N" terminus. Is this true for fish GPCRs? Have you tried a mammalian GPCR as a control?
Thanks Tom for your respone and sorry for delyaed reply. I have already started cloning in pmCherryC1. But my concern is that in this case the mCherry tag would be on the N-term and since the ligand binding site is also on the extracellular N-term of the GPCRs, this might interfere with the ligand binding.