School of Biological Sciences, University of the Punjab, Quaid-e-Azam Campus, Lahore, 54590, Pakistan.
Mol Biol Rep. 2013 Feb;40(2):983-97. doi: 10.1007/s11033-012-2140-8. Epub 2012 Nov 3.
Metallothioneins (MTs) are metal binding proteins that are rich in cysteine residues constituting 10-30 % of the total protein, and in which the thiol groups bind to the metal ions. The increasing amount of metal ions in the medium have shown increased production of MTs by different organisms such as bacteria, protozoa and mammals like humans. PMCd1 is the first gene ever discovered in Paramecium, a ciliated protozoan, that could produce this MT in response to cadmium. In this study the PMCd1syn gene has been cloned in pET41a expression vector and expressed in an Escherichia coli BL21-codonplus strain for the first time. Since the gene PMCd1 amplified from Paramecium contained 10 codons, which could act as stop codons during expression in E. coli, this gene of 612 bps was synthesized to substitute these (stop) codons for the Paramecium sp. specific amino acids. For stability of the expressed protein, glutathione-S-transferase gene was fused with PMCd1syn gene and coexpressed. The cells expressing PMCd1syn demonstrated increased accumulation of cadmium. This is the first report of cadmium MT protein expressed from Paramecium species, particularly from synthetic MT gene (PMCd1syn). This fusion protein, the molecular weight of which has been confirmed to be 53.03 kDa with MALDI analysis, is rich in cysteine residues, and has been shown for the first time in this ciliate to bind to and sequester Cd(2+)-ions.
金属硫蛋白（MTs）是富含半胱氨酸残基的金属结合蛋白，占总蛋白的 10-30%，其中巯基与金属离子结合。不同的生物体，如细菌、原生动物和哺乳动物（如人类），在介质中金属离子的含量增加，表明 MTs 的产量增加。PMCd1 是在草履虫中发现的第一个基因，它是一种纤毛原生动物，能够产生这种 MT 来应对镉。在这项研究中，PMCd1syn 基因已被克隆到 pET41a 表达载体中，并首次在大肠杆菌 BL21-codonplus 菌株中表达。由于从草履虫中扩增的 PMCd1 基因含有 10 个可能在大肠杆菌中表达时充当终止密码子的密码子，因此将该 612bp 的基因合成以替代草履虫特有的氨基酸。为了使表达的蛋白稳定，谷胱甘肽-S-转移酶基因与 PMCd1syn 基因融合并共同表达。表达 PMCd1syn 的细胞表现出镉的积累增加。这是首次从草履虫属中表达镉 MT 蛋白的报告，特别是从合成的 MT 基因（PMCd1syn）中表达。该融合蛋白的分子量已通过 MALDI 分析确认为 53.03kDa，富含半胱氨酸残基，并且首次在这种纤毛虫中显示与 Cd(2+)-离子结合并螯合。
