HIGH-LEVEL EXPRESSION IN ESCHERICHIA-COLI OF THE CATALYTICALLY ACTIVE FLAVIN DOMAIN OF CORN LEAF NADH-NITRATE REDUCTASE AND ITS COMPARISON TO HUMAN NADH-CYTOCHROME-B5 REDUCTASE

被引：52

作者：

HYDE, GE ^{[1
]}

CAMPBELL, WH ^{[1
]}

机构：

[1] MICHIGAN TECHNOL UNIV,DEPT BIOL SCI,HOUGHTON,MI 49931

来源：

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS | 1990年 / 168卷 / 03期

基金：

美国国家科学基金会;

关键词：

D O I：

10.1016/0006-291X(90)91168-R

中图分类号：

Q5 [生物化学]; Q7 [分子生物学];

学科分类号：

071010 ; 081704 ;

摘要：

Higher plant nitrate reductase can be divided into three functional domains representing its prosthetic groups: 1) flavin; 2) cytochrome b; and 3) Mo-pterin. The flavin domain has been synthesized by heterologous expression in Escherichia coli using a fragment of a corn leaf NADH:nitrate reductase cDNA clone, Zmnr1, which we had previously isolated and sequenced. A Xho2-BamH1 fragment was cut from Zmnr1, containing the sequence for the flavin domain, and ligated in the BamH1 site of expression vector pET3c. When this construct was expressed in E. coli, a 30 kD polypeptide was found to be newly synthesized. The flavin domain was purified to homogeneity using blue Sepharose and shown to have a molecular weight of 30 kD. The recombinant flavin domain has a ferricyanide reductase specific activity of 1000 μmol NADH oxidized/min/mg protein and a visible spectrum virtually identical to that of human NADH:cytochrome b5 reductase. © 1990.

引用

页码：1285 / 1291

页数：7

共 25 条

[1] CLONING OF DNA FRAGMENTS COMPLEMENTARY TO TOBACCO NITRATE REDUCTASE MESSENGER-RNA AND ENCODING EPITOPES COMMON TO THE NITRATE REDUCTASES FROM HIGHER-PLANTS [J].