Membrane proteins in dihybrid?

bram at mbcf.stjude.org bram at mbcf.stjude.org
Mon Dec 12 16:30:26 EST 1994


In article <witWo6q00iV5M8qEVn at andrew.cmu.edu>, "Terri G. Kinzy" <tk2h+ at andrew.cmu.edu> writes:
> During a recent discussion with someone intersted in using the dihybrid
> system to look for interacting proteins, the following question came up. 
> 
> Can you isolate clones for membrane associated or intermembrane proteins
> using this system.
> 
> Particularly, this person wants to find the receptor for his protein. 
> Has anyone tried this or looked specifically at pairs of proteins, one
> of which is presumed or known to be transmembrane.  I guess the
> questions are (1) transport into the nucleus, would these types of
> protein get stuck or (2) would they not be properly translated in the
> cytoplasm or unable to fold, etc..
> 
> Also, anyone tried truncating a putative or known transmembrane protein
> such that just the "soluble" domain is in the dihybrid system.
> 
> I look forward to interesting discussions of this question.
> 
> Terri Kinzy
> tk2h at andrew.cmu.edu
> Carnegie Mellon University
> 
I think it can work. I put the whole coding sequence (including
ER secretion signal sequence) of cyclophilin B in as bait, and
pulled out what looks like an integral membrane protein. Can't
swear to them interacting in mammalian cells, but clearly, including
the signal sequence fused to GAL4 DNA binding domain did not prevent
the fusion protein from working in the 2hybrid system

Rick Bram



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