The invention provides modification to the polynucleotide coding sequence
for Pichia angusta NAD (P)H: nitrate reductase [YNaR1; GenBank accession
number Z49110], which has Enzyme Commission number 1.7.1.2 (formerly EC
1.6.6.2), yielding the polynucleotide coding sequence for simplified
eukaryotic nitrate reductase (S-NaR1). The invention also provides a
method for recombinant expression of said polynucleotide code in the
cells of the methylotrophic yeast Pichia pastoris to produce the
polypeptide for S-NaR1, which binds the host-produced
molybdenum-molybdopterin cofactor and intracellularly forms catalytically
active, nitrate-reducing enzyme as small and stable multimeric proteins.
The invention also provides a method for rapid and high-yielding
purification of S-NaR1 by utilizing the hexa-histidine sequence at the
carboxyl-terminus of said polypeptide for immobilized metal affinity
chromatography.