Abstract
In plant cells, galactolipids are predominant, representing up to 50% of the lipid content in photosynthetic tissues. Galactolipid synthesis is initiated by MGDG synthases (MGDs), which use UDP-galactose as a donor sugar and diacylglycerol (DAG) as acceptor, to form monogalactosyldiacylglycerol (MGDG). This protocol is used to produce a recombinant form of Arabidopsis thaliana (A. thaliana) monogalactosyldiacylglycerol synthase 1 (MGD1) protein, in Escherichia coli (E. coli), using a two-step chromatographic purification procedure. The protein is easily expressed and purified to milligram quantities, suitable for biochemical and structural studies. The crystallization of MGD1 is also described.
Keywords: Photosynthetic tissues, Galactolipids, Monogalactosyldiacylglycerol, Crystallization, MGDG synthase
Background
Previous attempts to express plant MGDs in E. coli showed that approximately 99% of the recombinant protein accumulated in inclusion bodies (Miège et al., 1999). Solubilization of bacterial membranes using detergents, or in vitro inclusion bodies refolding protocols were developed and yielded pure and active fractions, sufficient to monitor the activity of the enzyme, but not to pursue its structural study (Nishiyama et al., 2003; Botté et al., 2005). Using a combination of different biochemical and biophysical techniques, and investigating the effects of various buffers and additives on the biochemical behavior of the enzyme, a simple, efficient and fast protocol was developed for the expression and purification of recombinant MGD1, addressing the problems frequently encountered with the purification of glycosyltransferases, particularly protein aggregation (Rocha et al., 2013). Conditions detailed here allowed the unprecedented production of a pure, soluble and active form of MGD1 and comply with both structural and functional dissections of this enzyme (Rocha et al., 2016). The protocol here described can also serve as a starting strategy to purify similar proteins.
Materials and Reagents
Equipment
Note: Cryocrystallography material for crystals manipulation in liquid nitrogen (LN2)
Procedure
Notes
Recipes
Acknowledgments
This protocol is an extension of that described in Rocha et al., 2013 and Rocha et al., 2016. This work was supported by Agence Nationale de la Recherche (grant ANR- 10-BLAN-1524, ReGal) and by the Rhône-Alpes region (France).
References
If you have any questions/comments about this protocol, you are highly recommended to post here. We will invite the authors of this protocol as well as some of its users to address your questions/comments. To make it easier for them to help you, you are encouraged to post your data including images for the troubleshooting.