Identification and characterization of a recombinant metallothionein protein from a marine alga, Fucus vesiculosus
Identification and characterization of a recombinant metallothionein protein from a marine alga, Fucus vesiculosus
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DOI：
10.1042/0264-6021:3380553
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发表时间：
1999-03-01
影响因子：
4.1
通讯作者：
Kille, P
中科院分区：
文献类型：
--
作者：
Morris, CA;Nicolaus, B;Kille, P
构建了适应长期高铜环境的大型藻类cDNA文库。为了研究金属硫蛋白（MT）基因存在的可能性，利用植物MT富含半胱氨酸基序设计的退化探针对文库进行筛选。该基因编码一个67个氨基酸的蛋白（1229 bp），具有MT蛋白的几个特征，包括16个半胱氨酸残基（24%）和一个芳香残基（1个）。虽然该蛋白序列与植物和无脊椎动物的MTs具有高度的一致性，但它在两个假定的金属结合结构域之间含有一个独特的“连接体”区域（14个氨基酸残基），不含半胱氨酸残基。这个延伸的连接子比原始脊椎动物MT中发现的三肽要大，但与植物MT序列中常见的40个氨基酸连接子也不一致。在大肠杆菌中表达的s肽Fucus MT融合蛋白的相对分子质量约为14 kDa。重组融合结合了7个Cd离子，其中50%在pH 4.1下解离。在厌氧条件下，Cd离子被Cu(I)取代，Cu(I)与蛋白质以13:1的比例结合。实验室暴露于高铜的水疱F.导致MT基因的诱导。本文首次从大型藻类中鉴定出铜暴露诱导的MT基因，该基因编码的蛋白产物可结合镉和铜。
A cDNA library was constructed from macroalgae adapted to prolonged elevated environmental copper levels. To investigate the possible existence of a metallothionein (MT) gene, the library was screened with degenerate probes designed using plant MT cysteine-rich motifs. A gene was identified (1229 bp) with a putative open reading frame (204 bp) encoding a 67-amino-acid protein exhibiting several characteristic features of MT proteins, including 16 cysteine residues (24%) and only one aromatic residue. Although the protein sequence showed high identity with plant and invertebrate MTs, it contained a unique 'linker' region (14 amino acid residues) between the two putative metal-binding domains which contained no cysteine residues. This extended linker is larger than the tripeptide found in archetypal vertebrate MTs, but does not conform either with the 40-amino-acid linkers commonly found in plant MT sequences. An S-peptide Fucus MT fusion protein expressed in Escherichia coli exhibited a relative molecular mass of similar to 14 kDa. The recombinant fusion bound seven Cd ions, of which 50% were dissociated at pH 4.1. Under anaerobic conditions, the Cd ions were displaced by Cu(I), which associated with the protein at a ratio of 13 : 1. Laboratory exposure of F. vesiculosus to elevated copper resulted in induction of the MT gene. Thus this paper describes, for the first time, an MT gene identified from macroalgae which is induced by copper exposure and whose encoded protein product binds cadmium and copper.