Park Hyun, Ahn In-Young, Choi Heeseon J, Pyo Sei Hong, Lee Hye Eun
Korea Polar Research Institute, Korea Ocean Research and Development Institute, Sa-2-Dong 1270, Ansan 426-744, South Korea.
Protein Expr Purif. 2007 Mar;52(1):82-8. doi: 10.1016/j.pep.2006.08.008. Epub 2006 Aug 24.
The genes for two apparent subtypes of metallothionein (MT) isoform were isolated from the Antarctic clam Laternula elliptica. Determination of the nucleotide sequence showed that the gene consists of 222 bp that code a 73-amino acid protein. The comparison between MT cDNA sequences of L. elliptica and other bivalves showed strong homologies on positions of cysteine residues, which are important for their metal binding abilities. The gene for the MT was inserted into a pET vector and overexpressed as a carboxyl terminal extension of glutathionein-S-transferase (GST) in Escherichia coli. After the GST fusion proteins had been purified by glutathione-Sepharose affinity chromatography column and digested with enterokinase, the MT was purified with gel filtration and analyzed for its biochemical properties. Recombinant MTs were reconstituted with Cd, Cu, and Zn, and kinetic studies of the reactions with electrophilic disulphide, DTNB, were investigated to explore their metal binding ability. It is revealed that the Cd-MT and Zn-MT react with DTNB biphasically, and that Zn-MT reacts with DTNB more rapidly, and with a significantly greater pseudo-first-order rate constant. Cu-MT reacts monophasically and releases metal slowly from MT.
从南极蛤类椭圆侧腕水母中分离出了金属硫蛋白（MT）亚型的两个明显基因。核苷酸序列测定表明，该基因由222个碱基对组成，编码一个73个氨基酸的蛋白质。椭圆侧腕水母与其他双壳贝类的MT cDNA序列比较显示，半胱氨酸残基位置具有很强的同源性，这些残基对它们的金属结合能力很重要。MT基因被插入到一个pET载体中，并在大肠杆菌中作为谷胱甘肽-S-转移酶（GST）的羧基末端延伸进行过表达。通过谷胱甘肽-琼脂糖亲和层析柱纯化GST融合蛋白并用肠激酶消化后，通过凝胶过滤纯化MT并分析其生化特性。用镉、铜和锌重构重组MT，并研究其与亲电二硫化物5,5'-二硫代双（2-硝基苯甲酸）（DTNB）反应的动力学，以探索它们的金属结合能力。结果表明，镉-MT和锌-MT与DTNB的反应呈双相性，且锌-MT与DTNB反应更快，假一级速率常数显著更大。铜-MT呈单相反应，金属从MT中缓慢释放。
