GFP in pombe

[S L Forsburg]

Nick Owen (nick_owen at wols.demon.co.uk)

> What do people generally use to get good signals in S.pombe
> 
> I intend to over express the fusion of my gene and GFP in something
> like pREP3x

We found that the original GFP was toxic when overproduced by REP3X
in pombe.  However, we get good results with a T65 mutant 
with modified codon usage (I don't recall which one offhand).   

Note that GFP is relatively small and will diffuse throughout the cell.
Also note that you need to ensure that your fusion is mimicking the
normal localization of your protein.  Often larger fusions do not
complement a mutnat, in which case you cannot be sure that the
protein is localizing properly (since it isn't working).

-susan
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S L Forsburg, PhD  forsburg at salk.edu
Molecular Biology and Virology Lab          
The Salk Institute, La Jolla CA 
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