protein-protein interaction and expression libraries.

DANIEL Y KIM dkim at nmsu.edu
Tue Feb 6 19:04:19 EST 1996


In article <jculi-0602962136250001 at powmod.cbm.uam.es> jculi at mvax.cbm.uam.es (Joaquim Culi ) writes:
>Dear netters,
>Do you know how to isolate proteins that interact with another known protein?
>I've been trying the two hybrid system but it hasn't worked very well
>because the piece of my protein that I suspect contains the interaction
>domain also activates transcription. Now I would like to screen a
>expression library in order to find proteins that interact with my protein
>but I don't know any protocol or even if it has been successfully done by
>someone else!!.
>
>Any protocol or reference will be greatly appreciated.
>
>Yours,
>
>Joaquim Culi


I am not sure where you're coming from, but these are my assumptions:

You have a protein which has been cloned.

You suspect that it interacts (physically binds to) another protein 
inside the cell

You do not know what the interacting protein or proteins are.

You want to have an isolate or clone of the interacting protein (s).

hmmmm

One way you might look into is to make a phage display cDNA library of 
the cell of interest.  That is, express all of the message in the cells 
as fusions onto the gene III coat protein of filamentous phage.

next, do an affinity-purification of any phage that are bound to 
immobilized known protein target.

Next, elute the phage and propagate them in E. coli.

You should now have an expression cDNA library that is enriched for cDNAs 
encoding proteins that bind to your known target.

I believe a recent BioTechniques described this process (November 1995 or 
thereabouts?).

My description is sketchy and may not be easy to follow (I am not at my 
best due to illness :(   ).  If you are interested, I might be able to 
dig up the citation.

Caveat:  This is a longshot.  By no means is it a standard method for 
isolating an interacting protein.  The usual method would be to use 
immunoprecipitation to isolate the binding protein, and make an antibody 
or degenerate oligonucleotide probe for screening a more conventional 
expression cDNA library.

Daniel Kim



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