Refolding and characterization of methionine adenosyltransferase from Euglena gracilis.
Protein Expr Purif
; 79(1): 128-36, 2011 Sep.
Article
em En
| MEDLINE
| ID: mdl-21605677
ABSTRACT
Methionine adenosyltransferase from Euglena gracilis (MATX) is a recently discovered member of the MAT family of proteins that synthesize S-adenosylmethionine. Heterologous overexpression of MATX in Escherichia coli rendered the protein mostly in inclusion bodies under all conditions tested. Therefore, a refolding and purification procedure from these aggregates was developed to characterize the enzyme. Maximal recovery was obtained using inclusion bodies devoid of extraneous proteins by washing under mild urea (2M) and detergent (5%) concentrations. Refolding was achieved in two steps following solubilization in the presence of Mg(2+); chaotrope dilution to <1M and dialysis under reducing conditions. Purified MATX is a homodimer that exhibits Michaelis kinetics with a V(max) of 1.46 µmol/min/mg and K(m) values of approximately 85 and 260 µM for methionine and ATP, respectively. The activity is dependent on Mg(2+) and K(+) ions, but is not stimulated by dimethylsulfoxide. MATX exhibits tripolyphosphatase activity that is stimulated in the presence of S-adenosylmethionine. Far-UV circular dichroism revealed ß-sheet and random coil as the main secondary structure elements of the protein. The high level of sequence conservation allowed construction of a structural model that preserved the main features of the MAT family, the major changes involving the N-terminal domain.
Texto completo:
1
Base de dados:
MEDLINE
Assunto principal:
Euglena gracilis
/
Redobramento de Proteína
/
Metionina Adenosiltransferase
Idioma:
En
Revista:
Protein Expr Purif
Assunto da revista:
BIOLOGIA MOLECULAR
Ano de publicação:
2011
Tipo de documento:
Article
País de afiliação:
Espanha