Expression, purification, and characterization of metallothionein-A from rainbow trout.
Recombinant metallothionein A (MT-A) from rainbow trout has been successfully produced in milligram quantities in Escherichia coli. cDNA has been subcloned into pGEX-6P.1 vector, in-frame with a sequence encoding an N-terminal glutathione-S-transferase (GST) tail. Purification to electrophoretic homogeneity has been obtained by affinity chromatography using GSH-Sepharose. After enzymatic cleavage of GST tail, the MT-A moiety shows a molecular weight, corresponding to the expected one (6630 Da). The final yield of the entire expression and purification process was about 5 mg of pure metallothionein per liter of bacterial culture. The effects of different reducing and alkylating agents have been evaluated at the level of the formation of higher molecular weight aggregates. To investigate the metal-binding ability of the recombinant MT-A, we carried out a spectrophotometrical titration with cadmium ions. Finally, we checked the metal dissociation by recording the UV absorbance of the protein as a function of the environmental pH.
Vergani
Laura
L
Department of Biophysical M & O Sciences and Technologies, Biophysical Division, University of Genova Medical School, Corso Europa 30, Genoa 16132, Italy. vergani@ibf.unige.it
Grattarola
Myriam
M
Dondero
Francesco
F
Viarengo
Aldo
A
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D002104:Cadmium / Q000378:metabolism; D003001:Cloning, Molecular; D018076:DNA, Complementary / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000378:metabolism; D005822:Genetic Vectors; D005982:Glutathione Transferase / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D008668:Metallothionein / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism; D008670:Metals; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D017686:Oncorhynchus mykiss; D010957:Plasmids / Q000378:metabolism; D011994:Recombinant Proteins / Q000737:chemistry; D013053:Spectrophotometry; D014466:Ultraviolet Rays
D018076:DNA, Complementary; D008670:Metals; D011994:Recombinant Proteins; D002104:Cadmium; D008668:Metallothionein; D005982:Glutathione Transferase