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Molecular cloning and expression of biologically-active diphtheria toxin receptor

Image Number 7 for United States Patent #5366874.

DT-resistant wild-type mouse L-M cells were co-transfected with a cDNA library constructed from RNA of highly toxin-sensitive monkey Vero cells and with a neomycin resistance gene. One DT-sensitive (DT.sup.S) colony was isolated from 8,000 stably-transfected neomycin-resistant L-M colonies screened by replica plate assays. The purified DT.sup.S mouse cells are highly toxin-sensitive (.about.1,000-fold more so than L-M cells and only .about.10-fold less than Vero cells) and are protected from DT toxicity on incubation with a nontoxic competitive DT inhibitor (CRM 197). Importantly, the cell surface receptors on DT.sup.S cells specifically bind radioiodinated DT, which is inhibited by unlabelled DT and the DT receptor-binding domain (HA6DT). A plasmid conferring DT-sensitivity (pDTS) was rescued from DT.sup.S cells, and the screening procedure repeated until a single cDNA encoding the DT receptor was isolated. The cDNA is predicted to encode an integral membrane protein that corresponds to the precursor of a human heparin-binding EGF-like growth factor. The DT sensitivity protein is thus a growth factor precursor which is exploited by DT, thus allowing the toxin to enter the cell.

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