Interesting GFP fusion protein problem with diffusion
'Mike' Michael J. Moser
moser at U.WASHINGTON.EDU
Mon Nov 22 16:46:29 EST 1999
Over the course of many transient GFP experiments in HT1080 fibrosarcoma
cells I see GFP in both the nucleus and the cytoplasm. GFP signal does
sometimes appear to be enriched within the nucleus in some cells,
suggesting as Sean proposes that there may be a cryptic NLS in GFP.
Mike Moser, Ph.D. Tel: 206-543-6585
UW Department of Pathology FAX: 206-543-3967
Box 357705 moser at u.washington.edu
Seattle, WA 98195 http://faculty.washington.edu/moser
On Sun, 21 Nov 1999, Ian Dawe wrote:
> 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
> Sorry I can't help you more, but it's interesting that this has been
> observed by someone else.
> In article <199911200254.UAA26802 at isua2.iastate.edu>, smurfdog at IASTATE.EDU
> > 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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