Expression, purification, and characterization of metallothionein-A from rainbow trout

Laura Vergani, Myriam Grattarola, Francesco Dondero, Aldo Viarengo

Risultato della ricerca: Contributo su rivistaArticolo in rivistapeer review

Abstract

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.

Lingua originaleInglese
pagine (da-a)338-345
Numero di pagine8
RivistaProtein Expression and Purification
Volume27
Numero di pubblicazione2
DOI
Stato di pubblicazionePubblicato - feb 2003

Fingerprint

Entra nei temi di ricerca di 'Expression, purification, and characterization of metallothionein-A from rainbow trout'. Insieme formano una fingerprint unica.

Cita questo