Expression, purification and characterization of beta domain and beta domain dimer of metallothionein.
In order to examine the independent self-assembly of the beta fragment of metallothionein and the interaction between two domains with the linker sequence, Lys-Lys-Ser, the chemically synthesized genes of the beta domain and its dimer (beta-KKS-beta) were cloned into vector pGEX-4T-1 and expressed as carboxyl terminal extension of glutathione-S-transferase (GST). After the GST fusion proteins had been digested with thrombin on a glutathione-Sepharose 4B affinity chromatography column, the beta domain and its dimer were purified with gel filtration and analyzed for their biochemical and spectroscopic properties. Amino acid composition and molecular mass are determined to be consistent with the expected value. The analysis of metal content shows that the beta domain and its dimer can bind with about 3eq and 6eq divalent metals, respectively. The characteristic peak presented around 254 nm in the UV and CD spectrum indicated that both the beta domain and its dimer are able to form the cadmium-thiolate clusters without the aid of the alpha domain. Furthermore, the absorption peak of the beta domain dimer is much higher than that of the beta domain, which suggested that there is an interaction between two beta domains. Finally, the metal-binding ability was determined by DTNB competitive reaction and the value of half dissociation pH, the results reveal that the beta domain dimer has stronger metal-binding ability than the single beta domain, which provides further evidence of the interaction between the two domains.
Zhou
Y
Y
National Laboratory of Protein Engineering, College of Life Sciences, Peking University, 100871, Beijing, PR China.
Li
L
L
Ru
B
B
D001483:Base Sequence; D002104:Cadmium / Q000737:chemistry; D002942:Circular Dichroism; D003001:Cloning, Molecular; D019281:Dimerization; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D015870:Gene Expression; D005822:Genetic Vectors; D008668:Metallothionein / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D008969:Molecular Sequence Data; D009154:Mutation; D013056:Spectrophotometry, Ultraviolet
D002104:Cadmium; D008668:Metallothionein