Enzymatic characterization of recombinant nitrate reductase expressed and purified from Neurospora crassa

Fungal Genet Biol. 2015 Jul:80:10-8. doi: 10.1016/j.fgb.2015.04.016. Epub 2015 Apr 23.

Abstract

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.

Keywords: Neurospora crassa; Nitrate reductase; Post-translational modification; Recombinant protein expression; Strep-tag®.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Antibodies, Monoclonal / chemistry
  • Escherichia coli
  • Gene Expression
  • Genetic Vectors
  • Mutation
  • Neurospora crassa / genetics*
  • Neurospora crassa / metabolism
  • Nitrate Reductase / chemistry
  • Nitrate Reductase / genetics*
  • Nitrate Reductase / isolation & purification*
  • Phosphorylation
  • Recombinant Proteins / genetics*
  • Recombinant Proteins / isolation & purification*

Substances

  • Antibodies, Monoclonal
  • Recombinant Proteins
  • Nitrate Reductase