JBC Ideal method for primary cell transfection

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J. Biol. Chem., Vol. 266, Issue 7, 4126-4130, Mar, 1991

Cloning, nucleotide sequence, and expression of the nitroreductase gene from Enterobacter cloacae

C Bryant, L Hubbard and WD McElroy
Department of Chemistry, University of California, San Diego, La Jolla 92093.

The "classical" nitroreductases of enteric bacteria are flavoproteins which catalyze the reduction of a variety of nitroaromatic compounds to metabolites which are highly toxic, mutagenic, or carcinogenic. The gene for the nitroreductase Enterobacter cloacae has now been cloned using an antibody specific to this protein. The nucleotide sequence of the structural gene and flanking regions are reported. Sequence analysis indicates that this gene belongs to a gene family of flavoproteins which have not been previously described. Analysis of the 5'-untranslated region reveals the presence of putative regulatory elements which may be involved in the modulation of the expression of this enzyme. The cloned gene was placed under the control of a T7 promoter for overexpression of the protein in Escherichia coli. The expressed recombinant protein was purified to homogeneity and exhibited physical, spectral, and catalytic properties identical to the protein isolated from E. cloacae.
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