problem:cleavage of fusion proteins

Julie Cullimore cullimor at toulouse.inra.fr
Thu Dec 6 04:36:08 EST 2001


We have been using the Clontech EGFP from the vector N2-EGFP to make 
C-terminal fusions with a variety of plasmamembrane located proteins, 
followed by Agrobacterium rhizogenes transformation to produce transgenic 
roots of Medicago truncatula (a plant similar to alfalfa).
In all cases (nine different constructs) the major GFP protein by Westerns 
is not the fusion but a protein similar in size to EGFP.
As this EGFP protein is either in the ER or vacuole (rather than the 
cytosol/nucleus - the normal destination of EGFP alone) it seems that the 
fusions are probably produced and target into the ER. However our fusions 
then appear to be degraded to leave mainly the GFP with varying levels of 
the fusion.

Have other persons had this problem with GFP fusions?
Is it particular to i) membrane targeting fusions ii) our plant, iii) the 
GFP and vector that I am using?

Many thanks for any help


Dr Julie Cullimore
Laboratoire de Biologie Moléculaire
des Relations Plantes-Microorganismes,
BP 27,
31326 Castanet-Tolosan Cedex,

Tel: 33 5 61 28 53 22 / 55 13
Fax: 33 5 61 28 50 61
Web site: http://www.toulouse.inra.fr/lbmrpm/eng/lbmrpm.htm
E-mail: cullimor at toulouse.inra.fr


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