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gain of function mutagenesis, how to do?

Frederik Börnke ricky_boernke at gmx.net
Wed Jan 19 04:24:09 EST 2000

Dear friends,

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.
I can think of basicly two strategies:
(1) Combine different domains of protein X from the two species in
order to find out which domain is responsible for the interaction
with isoform 2 of protein Y. Disadvantage: Time consuming, a lot
of cloning and no clue of the potential candidate domains.
(2) Performing random mutagenesis on the spinach protein and 
creating a library of mutagenized enzymes which is subsequently
screened against isoform 2 of protein Y in the yeast two hybrid
system. Advantage: No cloning, a large number of different mutations
can be screened in a single experiment. Disadvantage: If the 
interaction depends on larger structural changes than single 
amino acid substitutions they might not be covered by random 
And if random mutagenesis is the method of choice is it better to
use a PCR based protocol, or an E. coli mutator strain, such as 
Stratagene's XL-I red.
Any thoughts?



Frederik Boernke
Research Group of  Molecular Plant Physiology
Institute for Plant Genetics and Crop Plant Research (IPK)
Corrensstr. 3
06466 Gatersleben
Tel.  039482 -5 321
Fax. 039482 -5 515

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