School of Life Science, Shanxi University, Taiyuan 030006, PR China.
School of Life Science, Shanxi University, Taiyuan 030006, PR China; Wolfson College, University of Oxford, Oxford OX2 6UD, UK; Institute of Biomedical and Environmental Science & Technology, School of Life Sciences, Faculty of Creative Arts, Technologies and Science, University of Bedfordshire, University Square, Luton LU1 3JU, UK.
Ecotoxicol Environ Saf. 2021 Oct 1;222:112512. doi: 10.1016/j.ecoenv.2021.112512. Epub 2021 Jul 13.
Metallothioneins (MTs) are low molecular weight cysteine-rich proteins that bind to metals. Owing to their high cysteine (Cys) content, MTs are effective mediators of heavy metal detoxification. To enhance the heavy metal binding ability of MT from the freshwater crab Sinopotamon henanense (ShMT), sequence-based multiple sequence alignment (MSA) and structure-based molecular docking simulation (MDS) were conducted in order to identify amino acid residues that could be mutated to bolster such metal-binding activity. Site-directed mutagenesis was then used to modify the primary structure of ShMT, and the recombinant proteins were further enhanced using the SUMO fusion expression system to yield SUMO-ShMT1, SUMO-ShMT2, and SUMO-ShMT3 harboring one-, two-, and three- point mutations, respectively. The resultant modified proteins were primarily expressed in a soluble form and exhibited the ability to readily bind to heavy metals. Importantly, these modified proteins exhibited significantly enhanced heavy metal binding capacities, and they improved Cd, Cu and Zn tolerance and bioaccumulation in Escherichia coli (E. coli) in a manner dependent upon the number of introduced point mutations (SUMO-ShMT3 > SUMO-ShMT2 > SUMO-ShMT1 > SUMO-ShMT > control). Indeed, E. coli cells harboring the pET28a-SUMO-ShMT3 expression vector exhibited maximal Cd, Cu, and Zn bioaccumulation that was increased by 1.86 ± 0.02-, 1.71 ± 0.03-, and 2.13 ± 0.02-fold relative to that in E. coli harboring the pET28a-SUMO-ShMT vector. The present study offers a basis for the preparation of genetically engineered bacteria that are better able to bioaccumulate and tolerate heavy metals, thus providing a foundation for biological heavy metal water pollution treatment.
金属硫蛋白（MTs）是富含半胱氨酸的低分子量蛋白质，可与金属结合。由于其半胱氨酸（Cys）含量高，MTs 是重金属解毒的有效介质。为了提高来自河南华溪蟹（Sinopotamon henanense）的 MT（ShMT）的重金属结合能力，进行了基于序列的多重序列比对（MSA）和基于结构的分子对接模拟（MDS），以鉴定可突变以增强这种金属结合活性的氨基酸残基。然后，使用定点突变修饰 ShMT 的一级结构，并使用 SUMO 融合表达系统进一步增强重组蛋白，以产生分别带有一个、两个和三个点突变的 SUMO-ShMT1、SUMO-ShMT2 和 SUMO-ShMT3。所得修饰蛋白主要以可溶形式表达，并表现出与重金属结合的能力。重要的是，这些修饰蛋白表现出显著增强的重金属结合能力，并且它们以依赖于引入的点突变数的方式提高了大肠杆菌（E. coli）对 Cd、Cu 和 Zn 的耐受性和生物积累能力（SUMO-ShMT3> SUMO-ShMT2> SUMO-ShMT1> SUMO-ShMT>对照）。事实上，携带 pET28a-SUMO-ShMT3 表达载体的大肠杆菌细胞表现出最大的 Cd、Cu 和 Zn 生物积累，与携带 pET28a-SUMO-ShMT 载体的大肠杆菌相比，分别增加了 1.86±0.02、1.71±0.03 和 2.13±0.02 倍。本研究为制备更能生物积累和耐受重金属的基因工程菌提供了基础，为生物重金属水污染处理奠定了基础。
