Hi Shashi,

A lot of things can go wrong with fusion constructs and sometimes you just
have to try a bunch of different things. One thing is that the folding of
the fusion protein can result in weakend fluorescence because of
interaction between the GFP and your Dio3 protein or you may see altered
stability of the fusion protein. A couple of  different things we have
tried that made a difference were first to insert a short 12 to 15 amino
acid linker usually gly ser or gly ala that give a rod shaped structure to
separate the fusion protein from your protein.  The second thing is to try
another fusion protein. We have had good success with the mRFPmars
construct in situations were we could not get good fluorescence with a GFP
fusion construct. With some of our constructs were the amount of the
protein made was so low that we just could not see fluorescence from GFP
or RFP so we had to resort to using anti-GFP antibodies and a fluorescent
second antibody.

Best,
Daphne Blumberg


> Dear Dicty colleagues
> I have made an expression construct for Dictyostelium dio3 gene tagged
> to GFP at the N-terminal, using ptx-GFP vector. Dio3 has a
> stop codon TGA in the coding region that codes for Selenocycteine and
> needs 3' UTR secquence (SecIS) for translation and hence, the
> 3’ UTR sequence was engineered in the construct.
> I see some GFP fluorescence in the single cell stage, but the
> fluorescence disappears in multicellular structures. Dio3 is known to
> be expressed throughout growth and development. What could be the possible
> explanation for this? How should I prove the expression of fusion protein?
> Any suggestion will be appreciated.
>  
> Regards
>  
> Shashi Prakash Singh
> C/o, DR. R. Baskar
> Dept. of Biotechnology
> IIT Madras