Targeting of biotinylated compounds to its target tissue using a low-density lipoprotein receptor-avidin fusion protein.
The very high binding affinity of avidin to biotin is one of the highest to occur in nature. We constructed a fusion protein composed of avidin and the endocytotic LDL receptor in order to target biotinylated molecules to cells of the desired tissues. In addition to the native avidin, charge-mutated and nonglycosylated avidins were utilized as part of the fusion proteins, in order to modify its properties. All of the fusion protein versions retained the biotin-binding capacity. Although the specificity was not increased, however, fusion proteins composed of natural avidin and nonglycosylated avidin bound most efficiently to the biotinylated ligands. Fluorescence microscopy and atomic force microscopy studies revealed the expression of the fusion protein on cell membranes, and demonstrated specific and high-affinity binding of biotin to the low-density lipoprotein receptor (LDLR)-avidin fusion protein in vitro. Additionally, systemically administered biotinylated ligand targeted with high specificity the intracerebral tumors of rats that were expressing fusion protein after the virus-mediated gene transfer. These results suggest that local gene transfer of the fusion protein to target tissues may offer a novel tool for the delivery of biotinylated molecules in vitro and in vivo for therapeutic and imaging purposes.
A.I. Virtanen Institute, University of Kuopio, Kuopio, Finland.
P
P
Lehtolainen
T
T
Wirth
A K
AK
Taskinen
P
P
Lehenkari
O
O
Leppänen
M
M
Lappalainen
K
K
Pulkkanen
A
A
Marttila
V
V
Marjomäki
K J
KJ
Airenne
M
M
Horton
M S
MS
Kulomaa
S
S
Ylä-Herttuala
D000818:Animals; D001360:Avidin / Q000235:genetics*; D001710:Biotin / Q000378:metabolism; D015153:Blotting, Western / Q000379:methods; D001932:Brain Neoplasms / Q000628:therapy; D002458:Cell Fractionation; D002462:Cell Membrane / Q000378:metabolism; D018390:Gene Targeting; D015316:Genetic Therapy / Q000379:methods*; D005822:Genetic Vectors / Q000008:administration & dosage* / Q000235:genetics; D005910:Glioma / Q000628:therapy; D018625:Microscopy, Atomic Force; D008856:Microscopy, Fluorescence; D051381:Rats; D011973:Receptors, LDL / Q000235:genetics*; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism*; D012672:Semliki forest virus / Q000235:genetics
D011973:Receptors, LDL; D011993:Recombinant Fusion Proteins; D001360:Avidin; D001710:Biotin