Refolding and characterization of rat liver methionine adenosyltransferase from Escherichia coli inclusion bodies.
Methionine adenosyltransferase (MAT) catalyzes the synthesis of S-adenosylmethionine, the major methyl donor for transmethylation reactions. Attempts to perform structural studies using rat liver MAT have met with problems because the protein purified from cellular extracts is heterogeneous. Overexpression of the enzyme in Escherichia coli rendered most of the protein as inclusion bodies. These aggregates were purified by specific washes using urea and Triton X-100 and used for refolding. Maximal activity was obtained when chaotropic solubilization included the structural cation Mg(2+), the protein concentration was kept below 0.1 mg/ml, and denaturant removal was carried out in a two-step process, namely, a fast dilution followed by dialysis in the presence of 10 mM DTT or GSH/GSSG redox buffers. Refolding by this procedure generated the oligomeric forms, MAT I and III, which were basically indistinguishable from the purified rat liver forms in secondary structure and catalytic properties.
López-Vara
M C
MC
Instituto de Investigaciones Biomédicas Alberto Sols (CSIC), Arturo Duperier 4, Madrid, 28029, Spain.
Gasset
M
M
Pajares
M A
MA
D000818:Animals; D002850:Chromatography, Gel; D002942:Circular Dichroism; D004926:Escherichia coli / Q000201:enzymology*; D002479:Inclusion Bodies / Q000201:enzymology*; D007525:Isoelectric Focusing; D007700:Kinetics; D008099:Liver / Q000737:chemistry*; D008716:Methionine Adenosyltransferase / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism; D017510:Protein Folding; D017433:Protein Structure, Secondary; D051381:Rats; D011994:Recombinant Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism
D011994:Recombinant Proteins; D008716:Methionine Adenosyltransferase