Enzymatic characterization of recombinant nitrate reductase expressed and purified from Neurospora crassa.
We established an expression and purification procedure for recombinant protein production in Neurospora crassa (N. crassa). This Strep-tag® based system was successfully used for purifying recombinant N. crassa nitrate reductase (NR), whose enzymatic activity was compared to recombinant N. crassa NR purified from Escherichia coli. The purity of the two different NR preparations was similar but NR purified from N. crassa showed a significantly higher nitrate turnover rate. Two phosphorylation sites were identified for NR purified from the endogenous expression system. We conclude that homologous expression of N. crassa NR yields a higher active enzyme and propose that NR phosphorylation causes enhanced enzymatic activity.
Department of Plant Biology, Braunschweig University of Technology, 38106 Braunschweig, Germany.
Phillip
P
Ringel
Department of Plant Biology, Braunschweig University of Technology, 38106 Braunschweig, Germany.
Corinna
C
Probst
Department of Physical and Theoretical Chemistry and Braunschweig Integrated Center of Systems Biology (BRICS), Braunschweig University of Technology, 38106 Braunschweig, Germany.
Thorben
T
Dammeyer
Department of Physical and Theoretical Chemistry and Braunschweig Integrated Center of Systems Biology (BRICS), Braunschweig University of Technology, 38106 Braunschweig, Germany.
Sabine
S
Buchmeier
Cellular Proteomics, Helmholtz Centre for Infection Research, 38124 Braunschweig, Germany.
Lothar
L
Jänsch
Cellular Proteomics, Helmholtz Centre for Infection Research, 38124 Braunschweig, Germany.
Josef
J
Wissing
Department of Physical and Theoretical Chemistry and Braunschweig Integrated Center of Systems Biology (BRICS), Braunschweig University of Technology, 38106 Braunschweig, Germany.
Philip
P
Tinnefeld
Department of Plant Biology, Braunschweig University of Technology, 38106 Braunschweig, Germany.
Ralf R
RR
Mendel
Zoological Institute, Braunschweig University of Technology, 38106 Braunschweig, Germany.
Brigitte M
BM
Jockusch
Department of Plant Biology, Braunschweig University of Technology, 38106 Braunschweig, Germany. Electronic address: t.kruse@tu-bs.de.
Tobias
T
Kruse
D000911:Antibodies, Monoclonal / Q000737:chemistry; D004926:Escherichia coli; D015870:Gene Expression; D005822:Genetic Vectors; D009154:Mutation; D009492:Neurospora crassa / Q000235:genetics* / Q000378:metabolism; D050901:Nitrate Reductase / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification*; D010766:Phosphorylation; D011994:Recombinant Proteins / Q000235:genetics* / Q000302:isolation & purification*
D000911:Antibodies, Monoclonal; D011994:Recombinant Proteins; D050901:Nitrate Reductase
Neurospora crassa; Nitrate reductase; Post-translational modification; Recombinant protein expression; Strep-tag®