GFP fusion protein stability - linker region query?

Posted by Jennifer Clarke on
URL: http://confocal-microscopy-list.275.s1.nabble.com/GFP-fusion-protein-stability-linker-region-query-tp6867848.html

Dear list

We have a single cell cloned cell line which expresses eGFP fused to a G-protein coupled receptor in CHO cells, which we have been using for some time.

We always notice that after a few passages (after 2-3 weeks) the distribution of the GFP expression changes from being as expected associated with the receptor (cell surface and some intracellular compartments) to being diffuse and cytoplasmic, despite being maintained under the correct selection (geneticin).

I've heard anecdotal reports of similar apparent dissociation of GFP from the protein it is supposed to be fused to over similar time frames with other GFP-fusion proteins.

Is there any way to prevent this apparent dissociation?  Are there particular linker regions which one could choose in the design of fusion protein constructs which might be more resistant to such dissociation?

(The problem for us is it means we are unable to generate a stable-transfected cell line, derived from the parent eGFP-GPCR cell line, to express an addtional fusion protein with mKate (puramycin selection), as the eGFP expression is no longer associated with the GPCR over the time frame of generating a stable mKate-fusion protein expressing cell line in these cells, and we wish to investigate both fusion proteins in tandem.  The simple solution is to continue with working with transient mKate-fusion protein expression in the eGFP-GPCR cells, but we'd prefer to be able to generate a stable cell line expressing both correctly)

Kind regards
Jennifer

--
Jennifer Clarke BSc (Hons) PhD
Research Associate, Anatomy and Histology
Centre for Neuroscience, School of Medicine
&
Facility Manager, Optical Microscopy Suite, Flinders Microscopy

Flinders University
GPO Box 2100, Adelaide 5001
Phone: [phone] 8204 6637
Email: [hidden email]