Interesting GFP fusion protein problem with diffusion

smurfdog at IASTATE.EDU smurfdog at IASTATE.EDU
Fri Nov 19 22:02:16 EST 1999


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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