ICAAC abstracts..... I'm clueless, but it sounds cool, so here you are......
A Verotoxin B Subunit-Lambda Cro Chimeric Protein Defines a Novel Non-Viral
Gene Delivery System
L.M. FACCHINI, C.A. LINGWOOD
Hosp. for Sick Children Res. Inst. and Univ. of Toronto: Toronto, Ontario, Canada
Inefficient nuclear incorporation of foreign DNA remains a critical roadblock in the design of
effective, non-viral gene delivery systems. While most cells readily uptake exogenous DNA
delivered by traditional transfection, lipofection and electroporation protocols, the nucleic acids
remain within endosomal vesicles in all but a small percentage of cells. Verotoxin I (VT), an
AB5 subunit toxin produced by enterohaemorrhagic E. coli, binds to the glycolipid
globotriosylceramide (Gb3) on the cell surface and is internalized into pre-endosomes.
Following internalization, vesicular trafficking carries the VT binding (B) subunit to the Golgi,
endoplasmic reticulum and nucleus via a retrograde transport mechanism. To exploit this ability
of VT-B to bypass the barrier to nuclear transport, we fused the lambda Cro sequence-specific
DNA-binding protein to the carboxyl terminus of the non-toxic VT B subunit, generating a 14
kDa VTB-Cro chimeric protein. In electrophoretic mobility shift assays, VTB-Cro binds
specifically through the Cro domain to a 25 bp DNA fragment containing the consensus Cro
operator. In addition, VTB-Cro demonstrates a 2-28 fold increase in binding to a Gb3-coated
matrix compared with binding to an uncoated control matrix. Moreover, VTB-Cro binding to
Gb3 occurs simultaneously with DNA binding, allowing this fusion protein to target specific
DNA fragments to Gb3 positive cells. This novel VTB-Cro chimeric protein may find practical
applications in the fields of intracellular targeting, gene delivery and gene therapy. |
