title: Refolding from denatured inclusion bodies, purification to homogeneity and simplified assay of MGDG synthases from land plants.
abstract: In plant cells, the synthesis of monogalactosyldiacylglycerol (MGDG) is catalyzed within plastid envelope membranes by MGD proteins. MGDG synthesis was also reported in apicomplexan parasites, a phylum of protists harbouring a plastid that proved essential for the parasite survival. MGD activity is therefore a potent target for herbicidal and anti-parasitic molecules. In this study, we describe a detailed in vitro refolding protocol for denatured recombinant MGD accumulated in inclusion bodies from transformed Escherichia coli. The refolding process was dependent on CHAPS detergent and lipids, such as diacylglycerol and phosphatidylglycerol, as well as bivalent metals. Owing to this refolding procedure, the recombinant MGD protein from spinach was purified to homogeneity, allowing a definite characterization of its non-processivity and an investigation of its dimerization using cross-linking reagents. Additionally, using the portion of recombinant enzyme that accumulates in an active form in bacterial membranes, we developed a miniature assay for high-throughput screening for inhibitors.
authors.affiliation: UMR 5019 CNRS-CEA-INRA-Université Joseph Fourier, Laboratoire de Physiologie Cellulaire Végétale, Département de Recherche et Dynamique Cellulaire, CEA Grenoble, 17 rue des Martyrs, F-38054, 09, Grenoble Cedex, France.
authors.forename: Yoshitaka
authors.initials: Y
authors.lastname: Nishiyama
authors.forename: Hélène
authors.initials: H
authors.lastname: Hardré-Liénard
authors.forename: Stéphane
authors.initials: S
authors.lastname: Miras
authors.forename: Christine
authors.initials: C
authors.lastname: Miège
authors.forename: Maryse A
authors.initials: MA
authors.lastname: Block
authors.forename: Frédéric
authors.initials: F
authors.lastname: Revah
authors.forename: Jacques
authors.initials: J
authors.lastname: Joyard
authors.forename: Eric
authors.initials: E
authors.lastname: Maréchal
mesh_terms: D002850:Chromatography, Gel; D002852:Chromatography, Ion Exchange; D002855:Chromatography, Thin Layer / Q000379:methods; D003432:Cross-Linking Reagents / Q000737:chemistry; D004075:Diglycerides / Q000378:metabolism; D019281:Dimerization; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D005033:Ethylmaleimide / Q000494:pharmacology; D038983:Galactolipids / Q000032:analysis / Q000494:pharmacology; D005700:Galactosyltransferases / Q000096:biosynthesis / Q000737:chemistry* / Q000302:isolation & purification; D017353:Gene Deletion; D015870:Gene Expression; D005822:Genetic Vectors / Q000235:genetics; D006017:Glycolipids / Q000032:analysis; D006882:Hydroxyapatites / Q000737:chemistry; D002479:Inclusion Bodies / Q000737:chemistry; D007700:Kinetics; D008081:Liposomes / Q000378:metabolism; D008301:Maleimides / Q000737:chemistry; D010940:Plant Proteins / Q000096:biosynthesis / Q000737:chemistry*; D010944:Plants / Q000201:enzymology* / Q000235:genetics / Q000378:metabolism; D011489:Protein Denaturation; D017510:Protein Folding; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry* / Q000187:drug effects; D018724:Spinacia oleracea / Q000737:chemistry; D013696:Temperature; D014508:Urea / Q000737:chemistry; D014531:Uridine Diphosphate Galactose / Q000378:metabolism / Q000494:pharmacology
chemical_list: C011439:1,2-diacylglycerol; D003432:Cross-Linking Reagents; D004075:Diglycerides; D038983:Galactolipids; D006017:Glycolipids; D006882:Hydroxyapatites; D008081:Liposomes; D008301:Maleimides; D010940:Plant Proteins; D011994:Recombinant Proteins; C007388:digalactosyldiacylglycerol; C009909:monogalactosyldiacylglycerol; C013694:sulfoquinovosyl diglyceride; D014531:Uridine Diphosphate Galactose; C075594:1,6-bismaleimidohexane; D014508:Urea; D005700:Galactosyltransferases; C109990:1,2-diacylglycerol 3-beta-galactosyltransferase; D005033:Ethylmaleimide