GFP fusion protein stability - linker region query?

[Jennifer Clarke]

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]

[Vitaly Boyko]

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Hi Jennifer,
 
Please look at the description of the linker in the J. Virology paper (J. Virol.82(5):2405-17). It is the best linker for C-terminal fusions, GFP at C-terminus of the fussion construct. If you also need nt sequence of the linker, please let me know.
 
Vitaly


________________________________
From: Jen Clarke <[hidden email]>
To: [hidden email]
Sent: Thursday, October 6, 2011 9:03 PM
Subject: GFP fusion protein stability - linker region query?

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]

[Stefan Terjung]

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To join, leave or search the confocal microscopy listserv, go to:
http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
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Dear Listmembers,            

I believe this call for applications to visit advanced biological and
biomedical imaging facilities across Europe during the proof-of -concept
studies (FREE of charge) is very interesting to the members in Europe and
associated countries:
           
From: EIBIR Office [mailto:[hidden email]]
 Sent: Friday, September 30, 2011 3:32 PM
 Subject: Euro-BioImaging Proof-of-Concept Studies: Open Call for Users    
       

Dear Stefan Terjung,            

From January to July 2012 the large-scale pan-European research
infrastructure project Euro-BioImaging conducts a series of
proof-of-concept studies and therefore offers FREE access to advanced
biological and biomedical imaging facilities across Europe.            

The proof-of-concept studies will be a key pillar for the development of
eligibility criteria for future Euro-BioImaging nodes and specifically aim
to            

·        provide the opportunity for scientists to conduct their
research project using cutting edge imaging instruments            

·        test and refine standardized execution and access
protocols for future Euro-BioImaging facilities            

·        assess potential pitfalls for running these resources      
     

·        identify current community needs for access to different
imaging technologies            

Many European imaging facilities will serve as Euro-BioImaging
proof-of-concept study sites and will offer free access to a wide range of
cutting edge imaging technologies.            

The registration for users has recently opened and we invite you and
applicants from all over Europe to submit your research project proposals.  
         

Please forward this message to colleagues and friends, who are potentially
interested to participate as users in the Euro-BioImaging proof-of-concept
studies.            

Detailed information on the proof-of-concept studies and proposal
submission are provided at www.eurobioimaging.eu.            

We are looking forward to your active participation in this important stage
of the Euro-BioImaging project and remain with best wishes,            

Jan Ellenberg and Stefan Schönberg
 Scientific Coordinators of Euro-BioImaging            

sent by the project office        
--
__________________________________
Dr. Stefan Terjung
Advanced Light Microscopy Facility
EMBL Heidelberg
Meyerhofstr.1
D - 69117 Heidelberg
Phone [phone]
www.embl.de/almf
www.embl.org/elmi


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