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Just part of the CDS (like transmembrane domain) or the full-length CDS for prot

fusion protein FRET drug screen GPCR

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#1 Veevaa

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Posted 14 January 2014 - 02:03 AM

Hi, everyone, I got confused about strategy for the study of protein-protein interaction.

 

What I want to do is to make two fusion GPCRs with YFP and CFP, respectively,  and heterdimered with each other (if they can),  and next, i want use those cells transfected both plasmids to screen the compounds might have effects on the heterodimeration by FRET. I thought the plasmids should contain the full-length CDS sequence, but my boss told me that it needs only partrial CDS, like transmembrane domain, or the interaction domains coded by part of the CDS. If my boss was right, the fusion proteins lack some domain, can it be suitable for screening? Dose it make sense?

 

please anyone could help me

 

thanks



#2 dimensionsbio

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Posted 14 January 2014 - 03:56 PM

Hi Veevaa.  A single domain from a multi-domain protein can be expressed and used to study protein-protein interactions.  As long as you are expressing the domains that interact with each other, the interaction can still occur.



#3 Veevaa

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Posted 14 January 2014 - 05:39 PM

Hi Veevaa.  A single domain from a multi-domain protein can be expressed and used to study protein-protein interactions.  As long as you are expressing the domains that interact with each other, the interaction can still occur.

Many thanks, I still got a further question, a single domain of protein can be expressed to study protein-protein interactions, indeed, the protein-protein still occur, but the domain might lacks the site for compounds binding, is it suitble for screening?  

 

thanks



#4 dimensionsbio

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Posted 25 February 2014 - 04:30 AM

Hi Veevaa.  Sorry, just noticed your question.  The design of your experiment seems to require that you know if both the protein binding site and ligand (compound) binding site are present in your expression constructs.  In this case, searching the literature and comparing the sequence of other members of the protein family you are studying may provide you with some confidence that you have included both binding sites.  Another option is to make multiple constructs and test them out.

 

Hope this helps.







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