School of Life Science, Shanxi University, 92 Wucheng Road, Taiyuan, 030006, Shanxi, People's Republic of China.
Ecotoxicology. 2014 Jan;23(1):56-64. doi: 10.1007/s10646-013-1151-0. Epub 2013 Nov 27.
Metallothioneins (MTs) are ubiquitous metal-binding, cysteine-rich, small proteins and play a major role in metal homeostasis and/or detoxification in all organisms. In a previous study, a novel full length MT gene was isolated from the freshwater crab (Sinopotamon henanense), a species widely distributed in Shanxi and Henan Provinces, China. In this report, the gene for the crab MT was inserted into a PET-28a-6His-SUMO vector and recombinant soluble MT was over-expressed as fusions with SUMO in Escherichia coli. The recombinant fusion protein was purified by affinity chromatography and its biochemical properties were analyzed. In addition, on the basis of constructing SUMO-MT, two mutants, namely SUMO-MTt1 and SUMO-MTt2, were constructed to change the primary structure of SUMO-MT using site-directed mutagenesis techniques with the amino acid substitutions D3C and S37C in order to increase metal-binding capacity of MT. E. coli cells expressing SUMO-MT and these single-mutant proteins exhibited enhanced metal tolerance and higher accumulation of metal ions than control cells. The results showed that the bioaccumulation and tolerance of Zn(2+), Cu(2+) and Cd(2+) in these strains followed the decreasing order of SUMO-MTt1 > SUMO-MTt2 > SUMO-MT. E. coli cells have low tolerance and high accumulation towards cadmium compared to zinc and copper. These results show that the MT of S. henanense could enhance tolerance and accumulation of metal ions. Moreover, we were able to create a novel protein based on the crab MT to bind metal ions at high density and with high affinity. Therefore, SUMO-MT and its mutants can provide potential candidates for heavy metal bioremediation. This study could help further elucidate the mechanism of how the crab detoxifies heavy metals and provide a scientific basis for environment bioremediation of heavy metal pollution using the over-expression of the crab MT and mutant proteins.
金属硫蛋白（MTs）是一种普遍存在的、富含半胱氨酸的、能与金属结合的小蛋白，在所有生物中都在金属稳态和/或解毒中发挥主要作用。在之前的一项研究中，从中国山西省和河南省广泛分布的淡水蟹（Sinopotamon henanense）中分离出一种新型全长 MT 基因。在本报告中，将蟹 MT 基因插入 PET-28a-6His-SUMO 载体中，并在大肠杆菌中作为 SUMO 融合蛋白过表达重组可溶性 MT。通过亲和层析纯化重组融合蛋白，并分析其生化特性。此外，在构建 SUMO-MT 的基础上，通过定点突变技术构建了两种突变体，即 SUMO-MTt1 和 SUMO-MTt2，以改变 SUMO-MT 的一级结构，用 D3C 和 S37C 取代氨基酸，以提高 MT 的金属结合能力。表达 SUMO-MT 和这些单突变蛋白的大肠杆菌细胞表现出增强的金属耐受性和更高的金属离子积累，比对照细胞更高。结果表明，这些菌株对 Zn(2+)、Cu(2+)和 Cd(2+)的生物积累和耐受性遵循 SUMO-MTt1 > SUMO-MTt2 > SUMO-MT 的顺序。与锌和铜相比，大肠杆菌细胞对镉的耐受性低，积累高。这些结果表明，来自 S. henanense 的 MT 能够增强金属离子的耐受性和积累。此外，我们能够基于蟹 MT 构建一种新型蛋白质，以高密度和高亲和力结合金属离子。因此，SUMO-MT 及其突变体可以为重金属生物修复提供潜在的候选物。这项研究有助于进一步阐明蟹解毒重金属的机制，并为利用蟹 MT 和突变蛋白的过表达进行重金属污染环境的生物修复提供科学依据。
