title: Revisiting the expression and purification of MGD1, the major galactolipid synthase in Arabidopsis to establish a novel standard for biochemical and structural studies.
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.
authors.affiliation: CERMAV-CNRS, University of Grenoble 1, BP 53, F-38041 Grenoble cedex 9, France.
authors.forename: Joana
authors.initials: J
authors.lastname: Rocha
authors.forename: Magali
authors.initials: M
authors.lastname: Audry
authors.forename: Gaelle
authors.initials: G
authors.lastname: Pesce
authors.forename: Valérie
authors.initials: V
authors.lastname: Chazalet
authors.forename: Maryse A
authors.initials: MA
authors.lastname: Block
authors.forename: Eric
authors.initials: E
authors.lastname: Maréchal
authors.forename: Christelle
authors.initials: C
authors.lastname: Breton
mesh_terms: D017360:Arabidopsis / Q000201:enzymology* / Q000235:genetics*; D029681:Arabidopsis Proteins / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification* / Q000378:metabolism; D020134:Catalytic Domain; D003460:Crystallization; D004926:Escherichia coli / Q000166:cytology; D005700:Galactosyltransferases / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification* / Q000378:metabolism; D015870:Gene Expression; D005818:Genetic Engineering / Q000379:methods*
chemical_list: D029681:Arabidopsis Proteins; D005700:Galactosyltransferases; C528622:MGD1 protein, Arabidopsis