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GPCR expression in HEK293 - (Jul/19/2013 )

I am trying to express two fish GPCRs in HEK293 cells for their characterization. I have cloned the genes (2.5 kb each) in pTriEx3 and pmCherryN1 vectors. All the clones have been sequenced to confirm the presence of uninterrupted open reading frame. The problem is that I am unable to get protein expression from these constructs after transfection. I am able to red fluorescence in the empty pmCherry vector transfected cells, but there is absolutely no fluorescence in gene-pmCherry construct. The absence of protein and the presence of mRNA have been confirmed by western blotting and RT-PCR respectively, with appropriate positive and negative controls.

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 :)

-rmbio-

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?

-Tom M-

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.

-rmbio-