The invention provides modification to the polynucleotide coding sequence for <i>Pichia angusta</i> 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 <i>Pichia pastoris</i> 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.
申请公布号
WO03052063(A2)
申请公布日期
2003.06.26
申请号
WO2002US39341
申请日期
2002.12.10
申请人
THE NITRATE ELIMINATION COMPANY, INC.;CAMPBELL, WILBUR, H.;BARBIER, GUILLAUME, G.;CAMPBELL, ELLEN, R.
发明人
CAMPBELL, WILBUR, H.;BARBIER, GUILLAUME, G.;CAMPBELL, ELLEN, R.