Refolding and characterization of methionine adenosyltransferase from Euglena gracilis

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. Therefo...

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Bibliographic Details
Published in:Protein expression and purification 2011-09, Vol.79 (1), p.128-136
Main Authors: Garrido, Francisco, Estrela, Sylvie, Alves, Claudia, Sánchez-Pérez, Gabino F., Sillero, Antonio, Pajares, María A.
Format: Article
Language:English
Subjects:
Characterization
Cloning, Molecular
Escherichia coli
Escherichia coli - genetics
Euglena gracilis
Euglena gracilis - chemistry
Euglena gracilis - enzymology
Euglena gracilis - genetics
Gene Expression
Inclusion Bodies
Methionine adenosyltransferase
Methionine Adenosyltransferase - chemistry
Methionine Adenosyltransferase - genetics
Methionine Adenosyltransferase - isolation & purification
Methionine Adenosyltransferase - metabolism
Models, Molecular
Protein Multimerization
Protein Refolding
Protein Structure, Secondary
Refolding
Secondary structure
Solubility
Structural model
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Summary: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 (2 M) and detergent (5%) concentrations. Refolding was achieved in two steps following solubilization in the presence of Mg 2+; chaotrope dilution to
ISSN:1046-5928
1096-0279
DOI:10.1016/j.pep.2011.05.004