On 31 Mar 2000, B. Drees wrote:
> I am thinking about tagging some yeast proteins with GFP or GFP variants
> for localization. I'm trying to decide on the best strategy to use for
> this. My main questions are:
>> - Integrate tag into genome or express fusion on plasmid vector? So far
> my integrated tags are invisible so I am leaning toward using plasmids.
Keep in mind that GFP expressed at high levels tends to form clumps.
> - N-terminal or C-terminal fusions? Will an N-terminal fusion screw up
> localization signals?
It's likely, but there's no way to tell for sure, but the examples I've
seen are C-terminal. Some proteins have C-terminal localization signals,
so the ideal solution would be to try both. I think that you will
usually get cytoplasmic localization if the localization signal is screwed
up.
>> - Inducible or constitutive promoter on the vector?
If you use an inducible promoter, you might be able to control the
expression levels so that you don't wind up expressing too much and
getting clumps.
Also keep in mind that yeast strains which are ade2- (pink yeast) will
have green fluorescent vacuoles, because that pink adenine precursor
accumulates in the vacuole and is fluorescent.
Alex
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