公开(公告)号：US20060211084A1

公开(公告)日：2006-09-21

申请号：US10498697

申请日：2002-12-10

申请人： Wilbur Campbell ,  Guillaume Barbier ,  Ellen Campbell

发明人： Wilbur Campbell ,  Guillaume Barbier ,  Ellen Campbell

IPC分类号： C12N15/74 ,  C07H21/04 ,  C12P21/06 ,  C12N9/06 ,  C12N1/18

CPC分类号： C12Q1/26 ,  C12N9/0044 ,  C12N15/81 ,  G01N33/84 ,  G01N2333/906

摘要： 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.

摘要翻译： 本发明提供了对毕赤酵母NAD（P）H：硝酸还原酶[YNaR1; GenBank登录号Z49110]，其具有1.7.1.2的酶委员会（以前的EC 1.6.6.2），产生简化的真核硝酸还原酶（S-NaR1）的多核苷酸编码序列。 本发明还提供了一种用于在甲基营养酵母巴斯德毕赤酵母细胞中重组表达所述多核苷酸编码的方法，以产生用于S-NaR1的多肽，其结合宿主产生的钼钼卟吩辅因子并在细胞内形成催化活性的硝酸盐还原 酶作为小而稳定的多聚体蛋白。 本发明还提供了通过利用所述多肽的羧基末端的六组氨酸序列来固定金属亲和色谱来快速和高产纯化S-NaR1的方法。