The expression of a synthetic rainbow trout metallothionein gene in E. coli.
A synthetic gene for rainbow trout metallothionein was constructed and inserted into a dual origin plasmid where expression was induced by a temperature shift in a proteinase-deficient strain of Escherichia coli. The recombinant protein was purified to homogeneity, and a partial amino acid sequence was determined to confirm its identity. Its immunochemical characteristics were similar to those of native metallothionein from rainbow trout. The amounts of recombinant metallothionein produced were quantified in soluble cell extracts by ELISA. Low concentrations were detected when growth was performed either in L-broth or defined (GMM-II) medium. Supplementation of the medium with zinc or copper had no effect on the amount of metallothionein produced. By contrast, when cadmium was included in either L-broth or GMM-II medium, much higher concentrations of the protein within the cells (approx. 13 micrograms/mg soluble cell protein) were detected. This stabilisation of the protein by metal reconstitution in vivo is considered in relation to the selective uptake/exclusion of metals by the cells and its significance for the scavenging of certain precious or toxic heavy metals is discussed.
Kille
P
P
Department of Biochemistry, University of Wales College of Cardiff, U.K.
Stephens
P
P
Cryer
A
A
Kay
J
J
D000595:Amino Acid Sequence; D000818:Animals; D000936:Antigen-Antibody Complex; D001483:Base Sequence; D003001:Cloning, Molecular / Q000379:methods; D003429:Cross Reactions; D004926:Escherichia coli / Q000235:genetics* / Q000254:growth & development; D015870:Gene Expression; D005796:Genes; D005813:Genes, Synthetic; D007700:Kinetics; D008668:Metallothionein / Q000235:genetics* / Q000302:isolation & purification; D008670:Metals / Q000032:analysis; D008969:Molecular Sequence Data; D009838:Oligodeoxyribonucleotides / Q000138:chemical synthesis; D010957:Plasmids; D011994:Recombinant Proteins / Q000302:isolation & purification; D012487:Salmonidae / Q000235:genetics*; D012689:Sequence Homology, Nucleic Acid; D013054:Spectrophotometry, Atomic; D014337:Trout / Q000235:genetics*
D000936:Antigen-Antibody Complex; D008670:Metals; D009838:Oligodeoxyribonucleotides; D011994:Recombinant Proteins; D008668:Metallothionein