Protein Engineering, Vol. 13, No. 8, 575-581,
August 2000
© 2000 Oxford University Press
Characterization of diphtheria fusion proteins targeted to the human interleukin-3 receptor
Department of Cancer Biology, Wake Forest University School of Medicine, Winston-Salem, NC 27157, USA
Diphtheria fusion proteins are chimeric proteins consisting of the catalytic and translocation domains of diphtheria toxin (DT388) linked through an amide bond to one of a variety of peptide ligands. The ligand targets the molecule to cells and the toxin enters the cell, inactivates protein synthesis and induces cell death. Diphtheria fusion proteins directed to human myeloid leukemic blasts are a novel class of therapeutics for patients with chemotherapy refractory myeloid leukemia. Because of the presence of interleukin-3 (IL3) receptors on myeloid leukemic progenitors and its absence from mature myeloid cells, we synthesized four bacterial expression vectors encoding DT388 fused to human IL3. Different molecules were engineered to assess the effects of modifications on yield, purity and potency of product. The constructs differed in the size of the linker peptide between the DT388 and IL3 domains and in the presence or absence of an oligohistidine tag on the N- or C-terminus. Escherichia coli were transformed and recombinant protein induced and purified from inclusion bodies. Similar final yields of 3–6 mg of purified protein per liter of bacterial culture were obtained with each of the four molecules. Purity ranged from 70 to 90% after partial purification by anion-exchange, size-exclusion chromatography and/or nickel affinity chromatography. Proteins were soluble and stable at 4°C and –80°C in phosphate-buffered saline at 0.03–0.5 mg/ml. The fusion proteins showed predicted molecular weights by SDS–PAGE, HPLC and tandem mass spectrometry and had full ADP-ribosylating activities. Each was immunoreactive with antibodies to DT388 and IL3. Each of the fusion proteins with the exception of the one with an N-terminal oligohistidine tag showed full IL3 receptor binding affinity (Kd = 3 nM) and potent and selective cytotoxicity to IL3 receptor positive human myeloid leukemia cell lines (IC50 = 5–10 pM). In contrast, the N-terminal histidine-tagged fusion protein bound IL3 receptor with a 10-fold lower affinity and was 10-fold less cytotoxic to IL3 receptor positive blasts. Thus, we report a series of novel, biologically active DT388IL3 fusion proteins for potential therapy of patients with receptor positive myeloid leukemias.
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