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Expression, Purification and Crystallization of Recombinant Arabidopsis Monogalactosyldiacylglycerol Synthase (MGD1)   

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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: Photosynthethic 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.

Copyright: © 2016 The Authors; exclusive licensee Bio-protocol LLC.
How to cite: Rocha, J., Chazalet, V. and Breton, C. (2016). Expression, Purification and Crystallization of Recombinant Arabidopsis Monogalactosyldiacylglycerol Synthase (MGD1). Bio-protocol 6(24): e2064. DOI: 10.21769/BioProtoc.2064.
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