title: Expression of rat liver S-adenosylmethionine synthetase in Escherichia coli results in two active oligomeric forms.
abstract: A cDNA containing the complete coding sequence for rat liver S-adenosylmethionine synthetase was cloned into the prokaryotic expression vector pT7-7 and expressed in Escherichia coli BL21(DE3). A major additional band corresponding to a protein of 48 kDa was detected on SDS/PAGE after induction with isopropyl beta-D-thiogalactopyranoside. This protein was distributed in both the soluble and insoluble fractions and accounted for approx. 30% of the total bacterial protein. The soluble enzyme was fully active, as revealed by assays in vitro of S-adenosylmethionine synthetase activity. In addition, transformed bacteria exhibited highly increased levels of intracellular S-adenosylmethionine. Two active forms of the recombinant enzyme, with apparent molecular masses of 210 kDa and 110 kDa, were detected when cytosolic extracts of the transformed cells were fractionated by gel-filtration chromatography. It is concluded that the expressed S-adenosylmethionine synthetase polypeptide assemble as tetramers and dimers.
authors.affiliation: Instituto de Investigaciones Biomédicas, C.S.I.C., Madrid, Spain.
authors.forename: L
authors.initials: L
authors.lastname: Alvarez
authors.forename: J
authors.initials: J
authors.lastname: Mingorance
authors.forename: M A
authors.initials: MA
authors.lastname: Pajares
authors.forename: J M
authors.initials: JM
authors.lastname: Mato
mesh_terms: D000818:Animals; D001483:Base Sequence; D002850:Chromatography, Gel; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics*; D015870:Gene Expression; D018014:Gene Transfer Techniques; D007527:Isoenzymes / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification; D008099:Liver / Q000201:enzymology*; D046911:Macromolecular Substances; D008716:Methionine Adenosyltransferase / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction; D051381:Rats
chemical_list: D007527:Isoenzymes; D046911:Macromolecular Substances; D008716:Methionine Adenosyltransferase