title: Purification and characteristics of recombinant mouse metallothionein-I from Escherichia coli.
abstract: The mouse metallothionein-I (mMT-I) cDNA was amplified by polymerase chain reaction (PCR), inserted into vector pGEX-4T-1, and expressed in Escherichia coli as a carboxyl terminal extension of the 26-kDa glutathione-S-transferase (GST). Analyzed by SDS-PAGE, the amount of the expressed fusion protein GST-MT was over 50% of total cellular proteins. After the fusion protein had been digested with thrombin on a Glutathione-Sepharose 4B affinity chromatography column, recombinant mMT-I was purified by gel filtration on Sephadex G50. The results of molecular mass, amino acid composition and sequence of 10 amino acids at the N-terminus of the recombinant mMT-I demonstrate that the purified protein is the one we desired. The ratios of metal:protein and thiol:protein are the same as those of wild-type MT. The half-dissociation pHs of Cd, Cu, and Zn from recombinant mMT-I were 3.57, 1.40, and 5.20, respectively, which are in agreement with those from native rabbit MT-I. The ultraviolet absorbance and circular dichroism (CD) spectra at pH 8.0 and pH 2.0 were all similar to those of native MT, indicating that they have the same metal-thiolate structure even though six amino acid residues have been added at the N-terminus of the recombinant protein.
authors.affiliation: National Laboratory of Protein Engineering, College of Life Sciences, Peking University, Beijing, P.R. China.
authors.forename: Y
authors.initials: Y
authors.lastname: Xiong
authors.forename: B
authors.initials: B
authors.lastname: Ru
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D018076:DNA, Complementary / Q000096:biosynthesis*; D004926:Escherichia coli; D005982:Glutathione Transferase / Q000235:genetics*; D008668:Metallothionein / Q000235:genetics / Q000302:isolation & purification*; D051379:Mice; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction; D011994:Recombinant Proteins / Q000302:isolation & purification
chemical_list: D018076:DNA, Complementary; D011994:Recombinant Proteins; D008668:Metallothionein; D005982:Glutathione Transferase