Expression, purification and preliminary characterization of mussel (Mytilus galloprovincialis) metallothionein MT20.
Metallothioneins are rather ubiquitous metal-binding proteins induced by stressing or physiological stimuli. Two major metallothionein isoforms have been identified in mussel: MT10 and MT20. Nevertheless the high sequence homology, the two isoforms exhibit different expression and inducibility in vivo. We cloned and produced in Escherichia coli the MT20 isoform from Mytilus galloprovincilis. cDNA was subcloned into pGEX-6P.1 vector, in frame with a sequence encoding a glutathione-S-transferase (GST) tail. Recombinant protein was purified to electrophoretic homogeneity by affinity chromatography. After enzymatic cleavage of the GST tail the MT moiety was recovered with a final yield of about 5 mg of protein per litre of bacterial culture. The metal-binding ability of MT20 was assessed by absorption spectroscopy upon addition of cadmium equivalents and the metal release was checked as a function of the environment pH. Moreover the protein was analysed for the propensity to polymerization, typical of this class of protein, before and after exposure to reducing and alkylating agents.
Grattarola
Myriam
M
Department of Biophysical Sciences and Technologies M. & O (Di.S.T.Bi.M.O.), University of Genova, Largo Rosanna Benzi 10, 16132, Genova, Italy.
Carloni
Mara
M
Dondero
Francesco
F
Viarengo
Aldo
A
Vergani
Laura
L
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D002104:Cadmium / Q000378:metabolism; D003001:Cloning, Molecular; D019281:Dimerization; D015870:Gene Expression; D008668:Metallothionein / Q000235:genetics* / Q000302:isolation & purification* / Q000378:metabolism; D008969:Molecular Sequence Data; D049878:Mytilus / Q000235:genetics*; D010084:Oxidation-Reduction; D011485:Protein Binding; D017386:Sequence Homology, Amino Acid
D002104:Cadmium; D008668:Metallothionein