gain of function mutagenesis, how to do?

A.F. Simpson AFS7 at le.ac.uk
Wed Jan 19 15:09:29 EST 2000


Frederik Börnke wrote:
 
> I need some advice on the following experiment:
> Protein X from tobacco interacts with isoform 1 and 2 of protein Y
> in the yeast-two hybrid system. Protein X from spinach interacts
> with isoform 1 of protein Y only. X from tobacco and spinach share
> about 70% homology. Now I'd like to find out the structural basis
> of the different isoform specific interaction of the two proteins.

<snip choice between constructing chimeras and randomly mutating the
spinach protein>

Would there be any advantage of an inital set of experiments mutating
the tobacco protein X to look of sites which give _loss_ of function
(ie. produce a protein which interacts with isoform Y1 only)?

This might narrow down the important areas and allow you to target the
construction of chimeras to certain regions.

(I don't have much experience in this kind of experiment, so this is
more of a question than a suggestion.)

> Cheers
> Ricky

love
Anna




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