Increasing Expression of Neurospora crassa NADP+ -Dependent Glutamate Dehydrogenase

Helen Dunmore and Geoffrey Turner

Author address: 

. Department of Molecular Biology and Biotechnology, University of Sheffield, Sheffield, SIO 2UH, UK


NADP+-dependent glutamate dehydrogenases (GDH) found in many bacteria, plants and fungi catalyse the reductive amination of 2-oxoglutarate to L-glutamate which is an important step in the amino acid assimilation pathways. Work has been undertaken to increase the expression of the NADP+ dependent GDH protein, transcribed from the N. crassa am gene, as an aid to protein crystallisation studies of wt and mutant enzymes. The am132 deletion strain of N. crassa was transformed with the am gene coupled to the strong promoters grg-1, and also crp-2. Integration of the am gene occurred and the corresponding protein assays showed high enzyme levels, but over successive subcultures the quantity of GDH enzyme declined, possibly because the gene expression was being affected by methylation or quelling. The same result was obtained with both N. crassa promoters. Stabilisation only occurred at levels of expression lower than those produced by the N crassa wt. Expression of the am gene was also attempted in Aspergillus nidulans using the strong alcA promoter, but although levels of expression were obtained these were not higher than the N. crassa wt equivalent. In vitro mutagenesis is now in progress to investigate subunit interaction in this hexameric protein.

abstract No: 


Full conference title: 

    • ECFG 3rd (1996)