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Expression, purification, and characterization of histidine-tagged rat and human flavoenzyme dihydroorotate dehydrogenase

Bader, B ; Knecht, W LU ; Fries, M and Löffler, Monika (1998) In Protein Expression and Purification 13(3). p.414-422
Abstract

Mitochondrially bound dihydroorotate-ubiquinone oxidoreductase (dihydroorotate dehydrogenase, EC 1.3.99.11) catalyzes the fourth sequential step in the de novo synthesis of uridine monophosphate. Based on the recent functional expression of the complete rat dihydroorotate dehydrogenase by means of the baculovirus expression vector system in Trichoplusia ni cells, a procedure is described that allows the purification of baculovirus expressed enzyme protein fused to a carboxy-terminal tag of eight histidines. Extracts from mitochondria of Spodoptera frugiperda cells infected with the recombinant virus using Triton X-100 were loaded onto Ni2+-nitrilotriacetic acid agarose and histidine-tagged rat protein was selectively eluted with... (More)

Mitochondrially bound dihydroorotate-ubiquinone oxidoreductase (dihydroorotate dehydrogenase, EC 1.3.99.11) catalyzes the fourth sequential step in the de novo synthesis of uridine monophosphate. Based on the recent functional expression of the complete rat dihydroorotate dehydrogenase by means of the baculovirus expression vector system in Trichoplusia ni cells, a procedure is described that allows the purification of baculovirus expressed enzyme protein fused to a carboxy-terminal tag of eight histidines. Extracts from mitochondria of Spodoptera frugiperda cells infected with the recombinant virus using Triton X-100 were loaded onto Ni2+-nitrilotriacetic acid agarose and histidine-tagged rat protein was selectively eluted with imidazole-containing buffer. In view of our previously published work, the quality of the electrophoretic homogenous rat enzyme was markedly improved; specific activity was 130-150 micromol dihydroorotate/min per milligram; and the stoichiometry of flavin content was 0.8-1.1 mol/mol protein. Efforts to generate mammalian dihydroorotate dehydrogenases with low production costs from bacteria resulted in successful overexpression of the carboxy-terminal-modified rat and human dihydroorotate dehydrogenase in XL-1 Blue cells. By employing the metal chelate affinity chromatography under native conditions, the histidine-tagged human enzyme was purified with a specific activity of 150 micromol/min/mg and the rat enzyme with 83 micromol/min/mg, respectively, at pH 8.0-8.1 optimum. Kinetic constants of the recombinant histidine-tagged rat enzyme from bacteria (dihydroorotate, Km = 14.6 micromol electron acceptor decylubiquinone, Km = 9.5 micromol) were close to those reported for the enzyme from insect cells, with or without the affinity tag. HPLC analyses identified flavin mononucleotide as cofactor of the rat enzyme; UV-vis and fluorometric analyses verified a flavin/protein ratio of 0.8-1.1 mol/mol. By spectral analyses of the functional flavin with the native human enzyme, the interaction of the pharmacological inhibitors Leflunomide and Brequinar with their target could be clarified as interference with the transfer of electrons from the flavin to the quinone. The combination of the bacterial expression system and metal chelate affinity chomatography offers an improved means to purify large quantities of mammalian membrane-bound dihydroorotate dehydrogenases which, by several criteria, possesses the same functional activities as non-histidine-tagged recombinant enzymes.

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author
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publishing date
type
Contribution to journal
publication status
published
keywords
Animals, Baculoviridae/genetics, DNA, Complementary, Electrophoresis, Polyacrylamide Gel, Histidine/metabolism, Humans, Hydrogen-Ion Concentration, Kinetics, Molecular Sequence Data, Oxidoreductases/genetics, Oxidoreductases Acting on CH-CH Group Donors, Rats, Recombinant Proteins/genetics, Spodoptera
in
Protein Expression and Purification
volume
13
issue
3
pages
414 - 422
publisher
Academic Press
external identifiers
  • pmid:9693067
  • scopus:0032145981
ISSN
1046-5928
DOI
10.1006/prep.1998.0925
language
English
LU publication?
no
id
d38a76d0-13b4-4e30-9127-3a96a1f466e5
date added to LUP
2020-07-22 14:38:02
date last changed
2024-01-02 15:02:15
@article{d38a76d0-13b4-4e30-9127-3a96a1f466e5,
  abstract     = {{<p>Mitochondrially bound dihydroorotate-ubiquinone oxidoreductase (dihydroorotate dehydrogenase, EC 1.3.99.11) catalyzes the fourth sequential step in the de novo synthesis of uridine monophosphate. Based on the recent functional expression of the complete rat dihydroorotate dehydrogenase by means of the baculovirus expression vector system in Trichoplusia ni cells, a procedure is described that allows the purification of baculovirus expressed enzyme protein fused to a carboxy-terminal tag of eight histidines. Extracts from mitochondria of Spodoptera frugiperda cells infected with the recombinant virus using Triton X-100 were loaded onto Ni2+-nitrilotriacetic acid agarose and histidine-tagged rat protein was selectively eluted with imidazole-containing buffer. In view of our previously published work, the quality of the electrophoretic homogenous rat enzyme was markedly improved; specific activity was 130-150 micromol dihydroorotate/min per milligram; and the stoichiometry of flavin content was 0.8-1.1 mol/mol protein. Efforts to generate mammalian dihydroorotate dehydrogenases with low production costs from bacteria resulted in successful overexpression of the carboxy-terminal-modified rat and human dihydroorotate dehydrogenase in XL-1 Blue cells. By employing the metal chelate affinity chromatography under native conditions, the histidine-tagged human enzyme was purified with a specific activity of 150 micromol/min/mg and the rat enzyme with 83 micromol/min/mg, respectively, at pH 8.0-8.1 optimum. Kinetic constants of the recombinant histidine-tagged rat enzyme from bacteria (dihydroorotate, Km = 14.6 micromol electron acceptor decylubiquinone, Km = 9.5 micromol) were close to those reported for the enzyme from insect cells, with or without the affinity tag. HPLC analyses identified flavin mononucleotide as cofactor of the rat enzyme; UV-vis and fluorometric analyses verified a flavin/protein ratio of 0.8-1.1 mol/mol. By spectral analyses of the functional flavin with the native human enzyme, the interaction of the pharmacological inhibitors Leflunomide and Brequinar with their target could be clarified as interference with the transfer of electrons from the flavin to the quinone. The combination of the bacterial expression system and metal chelate affinity chomatography offers an improved means to purify large quantities of mammalian membrane-bound dihydroorotate dehydrogenases which, by several criteria, possesses the same functional activities as non-histidine-tagged recombinant enzymes.</p>}},
  author       = {{Bader, B and Knecht, W and Fries, M and Löffler, Monika}},
  issn         = {{1046-5928}},
  keywords     = {{Animals; Baculoviridae/genetics; DNA, Complementary; Electrophoresis, Polyacrylamide Gel; Histidine/metabolism; Humans; Hydrogen-Ion Concentration; Kinetics; Molecular Sequence Data; Oxidoreductases/genetics; Oxidoreductases Acting on CH-CH Group Donors; Rats; Recombinant Proteins/genetics; Spodoptera}},
  language     = {{eng}},
  number       = {{3}},
  pages        = {{414--422}},
  publisher    = {{Academic Press}},
  series       = {{Protein Expression and Purification}},
  title        = {{Expression, purification, and characterization of histidine-tagged rat and human flavoenzyme dihydroorotate dehydrogenase}},
  url          = {{http://dx.doi.org/10.1006/prep.1998.0925}},
  doi          = {{10.1006/prep.1998.0925}},
  volume       = {{13}},
  year         = {{1998}},
}