Newton D L, Nicholls P J, Rybak S M, Youle R J
Biochemistry Section, NINDS, National Institutes of Health, Bethesda, Maryland 20892.
J Biol Chem. 1994 Oct 28;269(43):26739-45.
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.
合成了人重组嗜酸性粒细胞衍生神经毒素（rEDN）的基因，并将其与编码针对人转铁蛋白受体的单链抗体（sFv）的基因融合（EDNsFv）。rEDN和EDNsFv在大肠杆菌BL21(DE3)的包涵体中均以不溶性蛋白形式表达。经过变性和复性后，通过肝素-琼脂糖层析对EDN和EDNsFv进行部分纯化。EDN的最终纯化通过Sephadex G-100实现，而含有6-组氨酸残基羧基末端的EDNsFv则使用金属螯合树脂Ni(2+)-次氮基三乙酸进行高度纯化。重组EDN具有与天然蛋白相似的核糖核酸酶活性，而融合蛋白的酶活性为天然EDN的6%-13%。融合蛋白能够与人转铁蛋白受体结合。与对人肿瘤细胞无固有细胞毒性的rEDN不同，EDNsFv融合蛋白对表达人转铁蛋白受体的人白血病细胞具有细胞毒性，IC50为0.2-1 nM。在1.3 nM的EDNsFv浓度下，对缺乏人转铁蛋白受体的细胞未观察到细胞毒性。针对人转铁蛋白受体的游离抗体E6可抑制EDNsFv的细胞毒性。人酶可通过与抗体融合来改造以获得细胞毒性特性。因此，它们可能是构建免疫融合蛋白的候选物，这些免疫融合蛋白可能比含有细菌或植物来源毒素部分的免疫毒素免疫原性更低。
