title: Expression of the Neurospora crassa metallothionein gene in Escherichia coli and its effect on heavy-metal uptake.
abstract: The gene coding for the Neurospora crassa metallothionein protein was chemically synthesized and cloned into the fusion expression vectors pMal-c and pMal-p. Cell-fractionation experiments demonstrated the proper localization of the pMal-c and pMal-p- expressed proteins to the cytosol and periplasm of the bacteria respectively. Control bacteria as well as the recombinant bacteria producing the metallothionein protein were incubated with solutions of 109Cd at concentrations of 0.2 microM, 1 microM, and 10 microM. The recombinant bacteria were able to accumulate significantly more 109Cd than control bacteria at all concentrations tested. Cadmium accumulation was rapid and highly selective. Maximum uptake was achieved at a pH of 7.0, with lower accumulation at lower or higher pH values. The pH-dependent uptake of cadmium by the recombinant bacteria was exploited to strip off the bound cadmium from the recombinant bacteria and to regenerate most of the cadmium-binding sites. These observations suggest the potential for using a metallothionein-based biosorbent for certain heavy-metal removal applications.
authors.affiliation: Center for Bio/Molecular Science and Engineering, Naval Research Laboratory, Washington, DC 20375, USA.
authors.forename: M
authors.initials: M
authors.lastname: Pazirandeh
authors.forename: L A
authors.initials: LA
authors.lastname: Chrisey
authors.forename: J M
authors.initials: JM
authors.lastname: Mauro
authors.forename: J R
authors.initials: JR
authors.lastname: Campbell
authors.forename: B P
authors.initials: BP
authors.lastname: Gaber
mesh_terms: D018528:ATP-Binding Cassette Transporters; D000595:Amino Acid Sequence; D001483:Base Sequence; D001692:Biological Transport; D002104:Cadmium / Q000378:metabolism*; D002352:Carrier Proteins / Q000235:genetics; D002451:Cell Compartmentation; D003600:Cytosol / Q000378:metabolism; D004274:DNA, Recombinant / Q000138:chemical synthesis; D004926:Escherichia coli / Q000378:metabolism*; D029968:Escherichia coli Proteins; D005656:Fungal Proteins / Q000235:genetics*; D005800:Genes, Fungal; D005813:Genes, Synthetic; D005822:Genetic Vectors; D058619:Maltose-Binding Proteins; D008668:Metallothionein / Q000096:biosynthesis / Q000235:genetics*; D008969:Molecular Sequence Data; D009004:Monosaccharide Transport Proteins; D009492:Neurospora crassa / Q000235:genetics*; D011993:Recombinant Fusion Proteins / Q000378:metabolism*; D014874:Water Pollutants, Chemical / Q000378:metabolism*
chemical_list: D018528:ATP-Binding Cassette Transporters; D002352:Carrier Proteins; D004274:DNA, Recombinant; D029968:Escherichia coli Proteins; D005656:Fungal Proteins; D058619:Maltose-Binding Proteins; D009004:Monosaccharide Transport Proteins; D011993:Recombinant Fusion Proteins; D014874:Water Pollutants, Chemical; C461599:maltose transport system, E coli; D002104:Cadmium; D008668:Metallothionein