Receptor characterization

[Mega [Andreas Stuermer]]

Hi everybody, 

I was wondering whether it is possible to characterize a specific receptor from an animal species.The human homologue of the receptor is known. 

So what would be needed is extract the receptor from the tissue, and analyze the structure. Is there a DIY way to do it? Is there anyone working on a similar project? How much would the equipment cost roughly? 

Would be very grateful for any keywords you might know!

[Matt Lawes]

Generally receptors are too low in expression to isolate directly in tissue. Make recombinant clone for over expression in E. coli, yeast, Pichia, baculovirus / insect cells etc.
Then you can purify & do crystal. Otherwise you can just map imputed amino acid sequence over existing crystal of homologous protein (I.e. in silico)

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[Nathan McCorkle]

I would read up on these keywords, and look for collaborators that you could get in with:

https://scholar.google.com/scholar?q=receptor%20characterization%20cryo%20em%20tomography

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-Nathan

[Bryan Jones]

If it's a membrane protein (since it's a receptor, I'm guessing it is), it is extra tricky to recombantly express at high levels, and to crystallize it. To add to that, without access to a high powered X-ray source and nice detector, DIY crystallography would be a no go. 

On the other hand, doing computational modeling of the receptor is definitely a realistic DIY project. You can use a service like Swiss Model (http://swissmodel.expasy.org/) to make some homology models of your animal receptors based upon the human receptor structure. 

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[Scott]

Andreas,

What specific species are you referring to? Generally people will clone the gene into an expression system to characterize it rather than try to purify the protein from tissue (very, very old school). Getting the cDNA for protein expression is often a challenge as you need the source RNA from the expressing tissue. Once you have the gene then you can focus on strategies to characterize it in what ever system you like.

Depending on your target species you might be able to get a Mammalian Gene Collection EST clone of your gene of interest.

http://genecollections.nci.nih.gov/MGC/

Harvard has some of the MGC EST with non-profit pricing (much lower than the commercial sources for the same clone).

https://plasmid.med.harvard.edu/PLASMID/Home.xhtml

Alternatively, you could send the sequence to a Gene Synthesis company if you have the money.

Cheers,

Scott

[Mega [Andreas Stuermer]]

Thanks everybody!! It's gonna take a while to dig through all the literature on the methods. 

For making a recombinant clone it would require finding the gene with old-school techniques, e.g. hybridisation probes I assume. Or maybe do chromosome walking on it? Seems like it's gonna be a lot of thought-work ;)