TY - JOUR
T1 - Expression, purification, and characterization of metallothionein-A from rainbow trout
AU - Vergani, Laura
AU - Grattarola, Myriam
AU - Dondero, Francesco
AU - Viarengo, Aldo
PY - 2003/2
Y1 - 2003/2
N2 - 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.
AB - 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.
UR - http://www.scopus.com/inward/record.url?scp=0037297681&partnerID=8YFLogxK
U2 - 10.1016/S1046-5928(02)00631-9
DO - 10.1016/S1046-5928(02)00631-9
M3 - Article
SN - 1046-5928
VL - 27
SP - 338
EP - 345
JO - Protein Expression and Purification
JF - Protein Expression and Purification
IS - 2
ER -