Characterization of diphtheria fusion proteins targeted to the human interleukin-3 receptor

A. E. Frankel, J. Ramage, M. Kiser, R. Alexander, G. Kucera, M. S. Miller

Research output: Contribution to journalArticle

57 Citations (Scopus)

Abstract

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 (K(d) = 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.

Original languageEnglish (US)
Pages (from-to)575-581
Number of pages7
JournalProtein Engineering
Volume13
Issue number8
StatePublished - 2000

Fingerprint

Interleukin-3 Receptors
Diphtheria
Fusion reactions
Proteins
Myeloid Leukemia
Interleukin-3
Myeloid Cells
Peptides
Molecules
Ligands
Administrative data processing
Diphtheria Toxin
Affinity chromatography
Recombinant proteins
Chemotherapy
Size exclusion chromatography
Inclusion Bodies
Cell death
Cytotoxicity
Tandem Mass Spectrometry

Keywords

  • Acute myeloid leukemia
  • Diphtheria toxin
  • Fusion protein
  • Interleukin-3

ASJC Scopus subject areas

  • Molecular Biology
  • Biochemistry

Cite this

Frankel, A. E., Ramage, J., Kiser, M., Alexander, R., Kucera, G., & Miller, M. S. (2000). Characterization of diphtheria fusion proteins targeted to the human interleukin-3 receptor. Protein Engineering, 13(8), 575-581.

Characterization of diphtheria fusion proteins targeted to the human interleukin-3 receptor. / Frankel, A. E.; Ramage, J.; Kiser, M.; Alexander, R.; Kucera, G.; Miller, M. S.

In: Protein Engineering, Vol. 13, No. 8, 2000, p. 575-581.

Research output: Contribution to journalArticle

Frankel, AE, Ramage, J, Kiser, M, Alexander, R, Kucera, G & Miller, MS 2000, 'Characterization of diphtheria fusion proteins targeted to the human interleukin-3 receptor', Protein Engineering, vol. 13, no. 8, pp. 575-581.
Frankel, A. E. ; Ramage, J. ; Kiser, M. ; Alexander, R. ; Kucera, G. ; Miller, M. S. / Characterization of diphtheria fusion proteins targeted to the human interleukin-3 receptor. In: Protein Engineering. 2000 ; Vol. 13, No. 8. pp. 575-581.
@article{b437aa53c8f84d21babd27774c845e84,
title = "Characterization of diphtheria fusion proteins targeted to the human interleukin-3 receptor",
abstract = "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 (K(d) = 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.",
keywords = "Acute myeloid leukemia, Diphtheria toxin, Fusion protein, Interleukin-3",
author = "Frankel, {A. E.} and J. Ramage and M. Kiser and R. Alexander and G. Kucera and Miller, {M. S.}",
year = "2000",
language = "English (US)",
volume = "13",
pages = "575--581",
journal = "Protein Engineering, Design and Selection",
issn = "1741-0126",
publisher = "Oxford University Press",
number = "8",

}

TY - JOUR

T1 - Characterization of diphtheria fusion proteins targeted to the human interleukin-3 receptor

AU - Frankel, A. E.

AU - Ramage, J.

AU - Kiser, M.

AU - Alexander, R.

AU - Kucera, G.

AU - Miller, M. S.

PY - 2000

Y1 - 2000

N2 - 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 (K(d) = 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.

AB - 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 (K(d) = 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.

KW - Acute myeloid leukemia

KW - Diphtheria toxin

KW - Fusion protein

KW - Interleukin-3

UR - http://www.scopus.com/inward/record.url?scp=0033833534&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=0033833534&partnerID=8YFLogxK

M3 - Article

VL - 13

SP - 575

EP - 581

JO - Protein Engineering, Design and Selection

JF - Protein Engineering, Design and Selection

SN - 1741-0126

IS - 8

ER -