Cloning, sequencing, and overexpression of a [2Fe-2S] ferredoxin gene from Escherichia coli.
Cloning, sequencing, and overexpression of a [2Fe-2S] ferredoxin gene from Escherichia coli.
复制标题
DOI：
10.1016/s0021-9258(19)49883-9
复制
发表时间：
1992-06
期刊：
影响因子：
--
通讯作者：
D. Ta;L. Vickery
中科院分区：
文献类型：
--
作者：
D. Ta;L. Vickery
大肠杆菌含有功能未知的可溶性[2Fe-2S]铁氧还蛋白（Knoell，H.- E、和Knappe，J.（1974）Eur. 50，245-252）。用纯化蛋白的抗血清筛选E. coli基因组表达文库中，我们克隆了编码该蛋白的基因（命名为FDX）。该基因的DNA序列预测在去除起始甲硫氨酸后的110个残基的多肽（多肽M（r）= 12，186，全蛋白M（r）= 12，358）。推导的氨基酸序列是惊人的相似，在动物线粒体中发现的铁氧还蛋白的功能与细胞色素P450酶和恶臭假单胞菌的铁氧还蛋白的功能与P450凸轮。当与人线粒体和恶臭假单胞菌铁氧化还原蛋白相比时，总体序列同一性约为36%，并且同一性包括提出用于协调铁簇的4个半胱氨酸残基。使用表达质粒，蛋白质过量产生约500倍，并且全蛋白以超过总细胞蛋白质的30%的量组装和积累。过表达的铁氧还蛋白表现出与动物铁氧还蛋白和恶臭假单胞菌铁氧还蛋白非常相似的吸收光谱、圆二色性和电子顺磁共振光谱。
Escherichia coli contains a soluble, [2Fe-2S] ferredoxin of unknown function (Knoell, H.-E., and Knappe, J. (1974) Eur. J. Biochem. 50, 245-252). Using antiserum to the purified protein to screen E. coli genomic expression libraries, we have cloned a gene (designated fdx) encoding this protein. The DNA sequence of the gene predicts a polypeptide of 110 residues after removal of the initiator methionine (polypeptide M(r) = 12,186, holoprotein M(r) = 12,358). The deduced amino acid sequence is strikingly similar to those of the ferredoxins found in animal mitochondria which function with cytochrome P450 enzymes and to the ferredoxin from Pseudomonas putida which functions with P450cam. The overall sequence identity is approximately 36% when compared with human mitochondrial and P. putida ferredoxins, and the identities include 4 cysteine residues proposed to coordinate the iron cluster. The protein was overproduced approximately 500-fold using an expression plasmid, and the holoprotein was assembled and accumulated in amounts exceeding 30% of the total cell protein. The overexpressed ferredoxin exhibits absorption, circular dichroism, and electron paramagnetic resonance spectra closely resembling those of the animal ferredoxins and P. putida ferredoxin.