Interesting GFP fusion protein problem with diffusion

Ian Dawe idawe at sfu.ca
Sun Nov 21 13:51:37 EST 1999


Hi Sean,

I've also observed when expressing GFP (in this case EGFP, 2-hr
destabilized from Clontech) in rat L6 myoblasts that there appears to be
some kind of localization. I don't examine the cells as carefully as I
probably should (I'm just using GFP as a reporter gene for promoter
analysis), but it appears to me that it's being localized in the cytoplasm,
sometimes even in very localized patches suggestive of vacuoles or even
crystals (as in polyhedrin, from baculovirus). Whether or not these aare
true occlusion bodies, I'm not sure. But GFP does seem to be undergoing
some sort of cellular localization.  Perhaps it has something to do with
the PEST degradation sequence fused with this variant to give it a 2-hr
half-life. 

Sorry I can't help you more, but it's interesting that this has been
observed by someone else.

Cheers,
Ian



In article <199911200254.UAA26802 at isua2.iastate.edu>, smurfdog at IASTATE.EDU
wrote:

> I've been doing subcellular localization studies using a GFP fusion 
> protein system.  I have created plasmids that encode different mutants 
> of my particular viral protein and inserted the mutants into the 
> EGFP-C2 vector and transiently transfected into a canine thymus cells.  
> In these experiments, some GFP-fusion proteins were found only in 
> the nucleus, whereas others were strictly cytoplasmic.  The finished 
> GFP-fusion protein is about 45 kDa and does not readily diffuse across 
> the nuclear membrane.
> 
> To determine if a smaller region within the mutant sequence of my 
> particular protein functions as a nuclear localization signal, I 
> inserted short oligonucleotides (corresponding to the region of 
> interest) into EGFP-C2 vector's multiple cloning site.  However, this 
> GFP-fusion appears to diffuse between cytosolic and nuclear spaces since 
> it smaller than the globular size limited by the nuclear pore complex .
> 
> MY QUESTIONS ARE:
> 
> 1) IS THERE ANY REPORT ON PRODUCTION OF A GFP VECTOR THAT 
> PRODUCES CYTOPLASMICALLY-LOCALIZED GFP?
> 
> 2) HAS ANYONE INSERTED A SECONDARY PROTEIN (i.e., beta-gal, etc.) 
> INTO THE GFP VECTOR TO PRODUCE A GFP-(b-gal)-(protein of interest) 
> TRIPLE FUSION THAT COULD NOT DIFFUSE ACROSS THE NUCLEAR 
> MEMBRANE?
> 
> It seems to me that either of these methods could limit diffusion and 
> allow testing of putative nuclear localization signals on GFP.  
> Answers and advice regarding either would be especially helpful.  
> 
> Thanks everyone!
> 
> Cheers,
> Sean Murphy
> Dept. of Vet. Micobiol. & Prevent. Med.
> Iowa State Univ.
> Ames, IA 50011
> smurfdog at iastate.edu
> 
> 
> 
> 
> 
> ---
> Sean C.  Murphy         
> Iowa State University, Ames, Iowa 50011
> 515.292.0924 (home) - 515.294.0900 (lab) - 515.294.8500 (fax)
> smurfdog at iastate.edu (e-mail)
> http://www.public.iastate.edu/~smurfdog (WWW)
> "Nature is the one place where miracles not only happen, but happen all 
> the time" - T. Wolfe




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