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GPCR [View Printable]
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Sandy
Group: Member
Posts: 117
Joined: Nov 23, 2004
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G protein coupled receptors are very interesting cell surface molecules. I have done several experiments trying to express the GPCR target we are working at in my lab. Cell lines I have chosen cannot express this molecule easily and the expression is lost or internalized quickly. Has anybody tried to express this molecule, which cell lines? |
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| Posted Dec 17, 2004, 1:15 AM |
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DD
Group: Member
Posts: 64
Joined: Dec 17, 2004
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GPCRs belong to a very big family of 7 transmemebrane domain. The recombinant cells expressing GPCR loose their expression very quickly. The best cells to use to my knowledge are: 293T transient cells. They cannot be stabilized but they express the protein very high for a short period time. |
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| Posted Dec 19, 2004, 19:06 PM |
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vanishing
Group: Member
Posts: 129
Joined: Apr 25, 2005
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which GPCR are you trying to express? |
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| Posted Apr 26, 2005, 0:49 AM |
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Sandy
Group: Member
Posts: 117
Joined: Nov 23, 2004
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| vanishing said: | | which GPCR are you trying to express? |
The GPCR molecule I am trying to express is an odorant receptor expressed on olfactory epithelial cells. It belongs to the class A of GPCRs based on Transmembrane homology. With the amino terminus outside and 7 transmembrane domains and the carboxy terminus inside. Thanks for helping. |
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| Posted Apr 26, 2005, 23:51 PM |
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MMatus
Group: Member
Posts: 5
Joined: May 06, 2005
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Hi! I was working whith the Mu and Delta opioid receptors that are CPCR's then they were expresed by CHO's cells.
There were no problems. |
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| Posted May 16, 2005, 19:38 PM |
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omid
Group: Member
Posts: 59
Joined: Dec 16, 2004
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| MMatus said: | Hi! I was working whith the Mu and Delta opioid receptors that are CPCR's then they were expresed by CHO's cells.
There were no problems. |
Most of people have problems expressing GPCRs. Did you ever used a transiant expression? I beleive the CHO cells are for stable transfection? |
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| Posted May 17, 2005, 20:45 PM |
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vanishing
Group: Member
Posts: 129
Joined: Apr 25, 2005
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Is there an antagonist to the receptor? Treating the cells in culture could inhibit internalization...
You could also think of trying an inducible expression system? (Tet-Off)? |
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| Posted May 18, 2005, 2:07 AM |
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MMatus
Group: Member
Posts: 5
Joined: May 06, 2005
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Hi again, we used the mu and delta GPCRs cloned at pCDNA3 plasmid, then it was transfected at CHOS cells. We have done some bioassays with those cultures. |
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| Posted May 19, 2005, 19:12 PM |
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stevenson
Group: Member
Posts: 1
Joined: May 26, 2005
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In general GPCRs are relatively not that difficult to express. I've been doing GPCR expression for more than ten years now and the more common mammalian cell lines I've used have been HEK293 cells, CHO-K1 for stable expression and COS-7 cells for transient. An optimized transfection protocol is key to a good protein expression system. It's also good to know how your ligand binds to the receptor in case there's a temperature/mechanical variable that you have to look out for. For large scale protein expression/production, the Sf9 insect cell line using baculoviruses has been particularly valuable for GPCR expression-not to mention the scalability! Good luck! |
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| Posted May 26, 2005, 17:14 PM |
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mottagui
Group: Member
Posts: 2
Joined: May 26, 2005
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May be I missed the point, but we have expressed numerous GPCRs in several different cell lines (HeLa, SK-NMc, HL60, Jutkat, Hek293 and U937) and they mostly expressed in a masureble manner (Ct= 29-32)
We clone them on pGBasic and are quite OK with working with them. But then , I have recently ventured into this area of biology, so some of you know much more than me.
Enlighten me!!
M |
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| Posted May 26, 2005, 15:35 PM |
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R
Group: Member
Posts: 1
Joined: May 27, 2005
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| Sandy said: | | G protein coupled receptors are very interesting cell surface molecules. I have done several experiments trying to express the GPCR target we are working at in my lab. Cell lines I have chosen cannot express this molecule easily and the expression is lost or internalized quickly. Has anybody tried to express this molecule, which cell lines? |
Have you checked eg: 1] J Biol Chem. 2005 Mar 25;280(12):11807-15. 2] Proc Natl Acad Sci U S A. 2004 Sep 14;101(37):13672-6. Where they [1] set up an assay system for odorant GPCRs in HeLa cells; or [2] improve cell surface delivery of odorant GPCR by coexpressing the receptor of interest with the beta2 adrenergic receptor. good luck R |
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| Posted May 27, 2005, 7:19 AM |
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Sandy
Group: Member
Posts: 117
Joined: Nov 23, 2004
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| stevenson said: | | In general GPCRs are relatively not that difficult to express. I've been doing GPCR expression for more than ten years now and the more common mammalian cell lines I've used have been HEK293 cells, CHO-K1 for stable expression and COS-7 cells for transient. An optimized transfection protocol is key to a good protein expression system. It's also good to know how your ligand binds to the receptor in case there's a temperature/mechanical variable that you have to look out for. For large scale protein expression/production, the Sf9 insect cell line using baculoviruses has been particularly valuable for GPCR expression-not to mention the scalability! Good luck! |
Thank you so much for your valuable input. We have tried many cell lines except the CHO-K1, we will try this cell line for the stable expression. |
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| Posted May 31, 2005, 17:49 PM |
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magali
Group: Member
Posts: 1
Joined: Jun 06, 2005
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Odorant receptors are often difficult to express, probably because they need special chaperones for proper folding. Why don't you look at these two papers:
Alexander A. Gimelbrant, Shannon L. Haley, and Timothy S. McClintock
Olfactory Receptor Trafficking Involves Conserved Regulatory Steps
J. Biol. Chem., Vol. 276, Issue 10, 7285-7290, March 9, 2001
(a special cell line that allows maturation of some of these receptors), and
J. Minic, M.-A. Persuy, E. Godel, J. Aioun, I. Connerton, R. Salesse, and E. Pajot-Augy
Functional expression of olfactory receptors in yeast and development of a bioassay for odorant screening
FEBS J., January 15, 2005; 272(2): 524 - 537.
(summarizes tricks that have worked with some difficult-to-express odorant receptors)
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| Posted Jun 07, 2005, 4:42 AM |
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roudi
Group: Member
Posts: 69
Joined: Dec 14, 2004
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| magali said: | Odorant receptors are often difficult to express, probably because they need special chaperones for proper folding. Why don't you look at these two papers:
Alexander A. Gimelbrant, Shannon L. Haley, and Timothy S. McClintock
Olfactory Receptor Trafficking Involves Conserved Regulatory Steps
J. Biol. Chem., Vol. 276, Issue 10, 7285-7290, March 9, 2001
(a special cell line that allows maturation of some of these receptors), and
J. Minic, M.-A. Persuy, E. Godel, J. Aioun, I. Connerton, R. Salesse, and E. Pajot-Augy
Functional expression of olfactory receptors in yeast and development of a bioassay for odorant screening
FEBS J., January 15, 2005; 272(2): 524 - 537.
(summarizes tricks that have worked with some difficult-to-express odorant receptors)
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In the recent work of Sayako et al. published in the journal of Neurochemistry, volume 90 issue 6 page 1453, September 2004, the importance of N-terminal glycosylation has been shown to be critical for OR expression and membrane trafficking. The C-terminal portion plays a role in defining proper conformation which specifies the G protain selectivity of the OR. Disruption of the N-terminal glycosylation site completely impairs its membrane trafficking. Functional expression of the mOR-EG was geatly enhanced by addition of extra-N terminal glycosylation sequences. |
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| Posted Oct 10, 2005, 18:46 PM |
Last edited Sep 08, 2008, 17:51 PM by frasermoss |
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Sandy
Group: Member
Posts: 117
Joined: Nov 23, 2004
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| stevenson said: | In general GPCRs are relatively not that difficult to express. I've been doing GPCR expression for more than ten years now and the more common mammalian cell lines I've used have been HEK293 cells, CHO-K1 for stable expression and COS-7 cells for transient. An optimized transfection protocol is key to a good protein expression system. It's also good to know how your ligand binds to the receptor in case there's a temperature/mechanical variable that you have to look out for. For large scale protein expression/production, the Sf9 insect cell line using baculoviruses has been particularly valuable for GPCR
expression-not to mention the scalability! Good luck! |
Could you please write your best protocol for a successful transient and stable transfection leading to accuratelty folded cell surface expressions?. I work with this odorant GPCR and I need to make monoclonal antibody against the cell surface protein. In most cases I only get internal exppression. |
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| Posted Oct 17, 2005, 18:54 PM |
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