A synthetic cadmium metallothionein gene (PMCd1syn) of Paramecium species: expression, purification and characteristics of metallothionein protein

Abstract

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.

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“…In our study, the results regarding concentration of metallothionein were comparable to those of Dar et al . (). Their group was interested in the production of metallothionein in various microorganisms in response to cadmium and they showed an increased accumulation of cadmium in cells expressing the protein metallothionein in comparison with control cells.…”

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“…It can chelate with heavy metal ions through sulfhydryl groups, so that microorganisms can maintain normal growth and metabolism in an environment rich in metal ions. Some studies have shown that the expression of bacterial metallothionein will be increased, and the overexpression of metallothionein also increases the proportion of heavy metal ions chelated under high concentrations of metal ions (Mathivanan et al 2021 ; Dar et al 2013 ).…”

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“…However, ciliate MTs present exclusive features with regard to classic MTs: they are longer proteins (60-193 aa) having higher cysteine content (22-54 residues) and aromatic amino acids are present in several of them 48 . So far, study of MTs in ciliates has been restricted to genera Tetrahymena 47,52 and Paramecium 56 . In Paramecium sp., MT gene is not well characterized.…”