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Revisiting the expression and purification of MGD1, the major galactolipid synthase in Arabidopsis to establish a novel standard for biochemical and structural studies
Authors:Joana Rocha  Magali Audry  Gaelle Pesce  Valérie Chazalet  Maryse A Block  Eric Maréchal  Christelle Breton
Institution:1. CERMAV-CNRS, University of Grenoble 1, BP 53, F-38041 Grenoble cedex 9, France;2. PCV, UMR 5168, CNRS-CEA-INRA-University of Grenoble 1, CEA-Grenoble, iRTSV, 17 rue des martyrs, 38054 Grenoble cedex 9, France
Abstract:Monogalactosyldiacylglycerol, the major lipid of plants and algal plastids, is synthesized by MGDG synthases (MGD). MGDs belong to the large glycosyltransferase family. They catalyze the transfer of a galactose residue from the donor UDP-Gal to a 1,2-sn-diacylglycerol acceptor. MGDs are monotopic proteins localized in the plastid envelope and, as such, they are difficult to purify. This study re-examined previous purification procedures and aimed to set up a standard protocol for expression and purification of recombinant MGD1, addressing problems frequently encountered with the purification of glycosyltransferases, particularly protein aggregation, and enabling crystallization for structural studies. Briefly, His-tagged versions of MGD1 were expressed in Escherichia coli and purified by a two-step procedure, including immobilized metal affinity chromatography and size-exclusion chromatography. We demonstrated that E. coli is an appropriate host cell to produce a soluble and active form of MGD1. We also investigated the effects of various buffers and additives used during the purification and concentration steps on the biochemical behavior of the enzyme. The protocol we developed typically yields milligram quantities of pure and homogenous protein material and proved suitable for crystallization and biochemical studies. We also revisited the conditions for activity tests and effects of known positive effectors of MGD1 such as phosphatidic acid and phosphatidylglycerol.
Keywords:Galactolipid  Galactolipid synthase  Phosphatidylglycerol  Phosphatidic acid
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