title: Expression and characterization of recombinant human eosinophil-derived neurotoxin and eosinophil-derived neurotoxin-anti-transferrin receptor sFv.
abstract: The gene for the human recombinant eosinophil-derived neurotoxin (rEDN) was synthesized and fused to the gene encoding a single chain antibody (sFv) to the human transferrin receptor (EDNsFv). Both rEDN and EDNsFv were expressed as insoluble proteins in inclusion bodies in Escherichia coli BL21(DE3). Following denaturation and renaturation, EDN and EDNsFv were partially purified by chromatography on heparin-Sepharose. Final purification of EDN was achieved by Sephadex G-100, whereas EDNsFv which contained a 6-histidyl residue carboxyl terminus was highly purified using the metal chelate resin, Ni(2+)-nitriloacetic acid. Whereas the recombinant EDN had ribonuclease activity that was similar to the native protein, the fusion protein had enzymatic activity that was 6-13% that of native EDN. The fusion protein was able to bind to the human transferrin receptor. In contrast to rEDN that had no inherent cytotoxicity to human tumor cells, the EDNsFv fusion protein was cytotoxic to human leukemia cells that express the human transferrin receptor with an IC50, 0.2-1 nM. At 1.3 nM EDNsFv, no cytotoxicity was observed on cells that lack the human transferrin receptor. Free antibody to the human transferrin receptor, E6, inhibited the cytotoxicity of the EDNsFv. Human enzymes may be engineered to acquire cytotoxic properties by fusing them to antibodies. Thus, they may be candidates for the construction of immunofusion proteins that may be less immunogenic than immunotoxins containing bacterial- or plant-derived toxin moieties.
authors.affiliation: Biochemistry Section, NINDS, National Institutes of Health, Bethesda, Maryland 20892.
authors.forename: D L
authors.initials: DL
authors.lastname: Newton
authors.forename: P J
authors.initials: PJ
authors.lastname: Nicholls
authors.forename: S M
authors.initials: SM
authors.lastname: Rybak
authors.forename: R J
authors.initials: RJ
authors.lastname: Youle
mesh_terms: D016475:3T3 Cells; D000595:Amino Acid Sequence; D000818:Animals; D000906:Antibodies; D001483:Base Sequence; D001667:Binding, Competitive; D004305:Dose-Response Relationship, Drug; D047208:Eosinophil-Derived Neurotoxin; D004804:Eosinophils / Q000737:chemistry / Q000201:enzymology; D004926:Escherichia coli / Q000235:genetics; D006801:Humans; D000922:Immunotoxins / Q000235:genetics / Q000378:metabolism / Q000494:pharmacology* / Q000633:toxicity; D004915:Leukemia, Erythroblastic, Acute / Q000188:drug therapy; D051379:Mice; D008969:Molecular Sequence Data; D009498:Neurotoxins / Q000235:genetics / Q000378:metabolism / Q000494:pharmacology*; D011485:Protein Binding; D011990:Receptors, Transferrin / Q000276:immunology*; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism / Q000494:pharmacology* / Q000633:toxicity; D012260:Ribonucleases; D057127:Single-Chain Antibodies
chemical_list: D000906:Antibodies; C090089:EDNsFv anti-transferrin receptor; D000922:Immunotoxins; D009498:Neurotoxins; D011990:Receptors, Transferrin; D011993:Recombinant Fusion Proteins; D057127:Single-Chain Antibodies; D047208:Eosinophil-Derived Neurotoxin; D012260:Ribonucleases