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:
>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
>Any protocol or reference will be greatly appreciated.
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).
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
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.
More information about the Methods