title: 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.
authors.affiliation: School of Biological Sciences, University of the Punjab, Quaid-e-Azam Campus, Lahore, 54590, Pakistan.
authors.forename: Saira
authors.initials: S
authors.lastname: Dar
authors.forename: Rukhsana N
authors.initials: RN
authors.lastname: Shuja
authors.forename: Abdul Rauf
authors.initials: AR
authors.lastname: Shakoori
mesh_terms: D000595:Amino Acid Sequence; D001483:Base Sequence; D002104:Cadmium / Q000737:chemistry* / Q000378:metabolism; D002846:Chromatography, Affinity; D003001:Cloning, Molecular; D004926:Escherichia coli; D015870:Gene Expression; D005813:Genes, Synthetic; D002479:Inclusion Bodies / Q000378:metabolism; D008668:Metallothionein / Q000096:biosynthesis / Q000235:genetics* / Q000302:isolation & purification; D008958:Models, Molecular; D008969:Molecular Sequence Data; D010247:Paramecium / Q000235:genetics*; D010446:Peptide Fragments / Q000737:chemistry; D011485:Protein Binding; D015202:Protein Engineering; D015800:Protozoan Proteins / Q000096:biosynthesis / Q000235:genetics* / Q000302:isolation & purification; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000235:genetics / Q000302:isolation & purification; D020539:Sequence Analysis, Protein; D040681:Structural Homology, Protein
chemical_list: D010446:Peptide Fragments; D015800:Protozoan Proteins; D011993:Recombinant Fusion Proteins; D002104:Cadmium; D008668:Metallothionein