Expression from Gal10-protmoter

Tomas Drgon DRGON at fns.uniba.sk
Mon Feb 21 08:48:20 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...)
>
> *******************************
> 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...."
> *******************************

Hi Hannes.

Are you sure your gene is transcribed?

I have got similar problems with the mammalian
ADP/ATP translocator expression in yeast (from Gal1).

Regards

Tomas Drgon



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