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
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.
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)
"Nature is the one place where miracles not only happen, but happen all
the time" - T. Wolfe