Construction, Expression And Characterization Of The Fusion Protein DAB₃₈₉-(GLy₄Ser)₂-EGF And Its Specific Cytotoxic Activity

Diphtheria toxin(DT) is a single chain exotoxin which consists of 535 amino acids, secreted by corynebacterium diphtheriae carrying DT gene. Eukaryotic cells are very sensitive to DT, only one or two molecules of DT can kill a cell. The mechanism by which DT causes a cell death is that once DT enters the organism, the complete DT molecule is cleaved into A and B fragments at residue 192Arg/193Arg, the receptor binding domain of the fragment B will bind to DT receptor, and the transmembrane domain of the fragment B will translocate the fragment A into the cytoplasm of the sensitive cells, and the fragment A in the cytoplasm catalyses the transfer of the ADP-rebosylation group of NAD +to elongation factor-2(EF-2), as a consequence, EF-2 is inactivated, and the protein synthesis of the sensitive cell is held back.Epidermal growth factor (EGF)is a 53-amino acid single chain polypeptide which stimulates cellular proliferation upon interaction with specific receptors expressed on cells of epidermal and mesenchymal origin.The EGF receptor is a glycoprotein composed of an extracellular ligand binding domain,a membrane-spanning region and an inner domain possessing intrinsic protein tyrosine kinase activity. Transforming growth factor- a (TGF-a ), a molecule structurally related to EGF and secreted by various tumor tissues, is also a ligand for the EGF receptor. Several findings raise the possibility that the EGF receptor plays a role in induction or maintenance of malignant disease. Enhanced expression of the EGF receptor has been described in a wide variety of malignancies including esophageal, lung, thyroid, gastric, bladder, renal, prostate, ovarian, endometrial, cervical, breast, and brain. For example, the number of EGF receptors is increased as much as 500-fold in lung carcinomas compared to adjacent normal tissue. In some tumors, such as glioblastoma, elevated EGF receptor expression occurs by amplification of the EGF receptor gene resulting in up to a 60-fold increase in the number of gene copies.To therapeutically target the cytotoxic action of diphtheria toxin to EGF receptor expressing tumor cells, We have constructed two fusion gene in which the sequences for the binding domain of diphtheria toxin have been replaced by those for human EGF or linker-EGF respectively. Protein were expressed successfully, accounting for 23%of the total bacterial protein. The results of the SDS-PAGE and the western-bloting stained that there was a band about 50,000 dalton agree with predicted result. The resultsshow that fusion toxins.DAB389EGF and DAB389-L-EGF,bind specifically to the EGF receptor and inhibit protein synthesis in a variety of EGF receptor expressing human tumor cell lines with an IC50 as low as 0.1 pM.DAB389EGF and DAB389-L-EGF were purified by a four-step purification scheme which included hydrophobic interaction chromatography, anion exchange, dye affinity and gel filtration chromatography. The purities of the final DAB389EGF and DAB389-L-EGF preparations were 89% and >95%, respectively,as estimated by Dual-wavelength densitometry scanning of Coomassie Blue Stained SDS-PAGE. Comparisons of DAB3g9EGF and DAB389-L-EGF showed that DAB389-L-EGF was consistently 3 to 5 fold more cytotoxic than DAB389EGF.Cytotoxicity of DAB389-L-EGF and DAB3g9-EGF were tested on 7 kinds of human tumor cell lines by XTT method, which have been reported to express high numbers of EGF receptors, for example: lung carcinoma(A549),Oral carcinoma(KB),Cervical carcinoma (Hela), Pharynx carcinoma (HeP-2), hepatocellular carcinoma(HePG-2), Breast carcinoma(MDA), these cell lines were extremely sensitive to DAB389-L-EGF; and other 4 kinds of normal tissue cell lines were insensitive to DAB389-L-EGF.