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Expression from Gal10-protmoter

Hannes Klump HANNES at bch.univie.ac.at
Fri Feb 18 14:57:28 EST 1994


Hi, Buddies!
At current I am trying to express a viral proteinase in yeast. For
this purpose I cloned the coding sequence of the proteinase with
the Gal1-10 promotor into yiplac204 (Gietz and Sugino,
Gene74:527,1988) so that expression is directed from the Gal10 part
of the promoter.
After integrating this thing into the trp-locus I tried to detect my
protein after indduction via Western-blot:
After growing the cells in 2% Raffinose till they were in log-phase I
started the induction with 2% Galactose and took samples every quarter
hour up to 4h.
The crux I have now is that I am not able to detect my protein with
my antiserum on a Western blot:
is it possible that the protein level in a cell having just one or
two copies of the construct in the genome is not high enough albeit
using the Gal-promoter for expression?
What is the avarage protein level one could expect using this
expression-system?

The second problem is that the background of the western is quite high
because the rabbit immunized with my protein seems to have 'seen'
yeast somewhere before...
I've tried reducing the background by preadsorbing the antiserum with
blotted yeast-lysate without the construct; it didn't work that well
as I expected. Does anyone of you have a good protocol for this
purpose?

I have to apologize for my quite ignorant questions but I am just a
yeast-newcomer trying to establish this organism as a model system in
our virological lab. (And it's not easy to convince old-established
virologists who work with HeLa and Co. that Yeast could be a great
model-organism for some fundamental biological questions...)










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Hannes Klump
Institute of Biochemistry
University of Vienna
Dr. Bohrgasse 9/3
A-1030 Vienna / Austria
e-mail: Hannes at bch.univie.ac.at
"Trust me, I'm a Scientist...."
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