Targeting of green fluorescent protein expression to the cell surface.
We have previously reported on GPI-anchored fusion proteins that bind radioactive isotopes. We targeted their expression to the cell surface to obtain a marker protein detectable by nuclear and optical imaging (1, 2). Here we suggest a novel approach for targeting a model protein (GFP) to the exoplasmic surface of the plasma membrane. An expression vector (pcPEP-GFP) was constructed containing GFP cDNA fused with the fragment encoding the N-terminal cytoplasmic domain and signal peptide/membrane anchoring domain of the rabbit neutral endopeptidase (PEP-GFP). Flow cytometry showed green fluorescence in 45% of cells transfected with GFP and in 34% of cells transfected with PEP-GFP (24 h after transfection). Fluorescence microscopy of fixed cells stained with rhodaminated anti-GFP antibodies showed positive reaction only in the case of PEP-GFP-transfected cells indicating cell-surface expression. The PEP-GFP fusion protein was identified as a component of the light microsomal and Golgi fractions by immunoblotting.
Simonova
M
M
Center for Molecular Imaging Research, Massachusetts General Hospital, Building 149, 13th Street, Charlestown, Massachusetts, 02129, USA.
Weissleder
R
R
Sergeyev
N
N
Vilissova
N
N
Bogdanov
A
A
D000818:Animals; D019556:COS Cells; D002458:Cell Fractionation; D002462:Cell Membrane / Q000378:metabolism*; D018076:DNA, Complementary; D005434:Flow Cytometry; D017930:Genes, Reporter; D049452:Green Fluorescent Proteins; D007700:Kinetics; D008164:Luminescent Proteins / Q000096:biosynthesis / Q000235:genetics*; D015260:Neprilysin / Q000096:biosynthesis / Q000235:genetics; D021382:Protein Sorting Signals / Q000096:biosynthesis / Q000235:genetics; D011817:Rabbits; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis; D014162:Transfection / Q000379:methods
D018076:DNA, Complementary; D008164:Luminescent Proteins; D021382:Protein Sorting Signals; D011993:Recombinant Fusion Proteins; D049452:Green Fluorescent Proteins; D015260:Neprilysin