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Expression and Purification of DGD2, a Chloroplast Outer Membrane-Associated Glycosyltransferase for Galactolipid Synthesis
journal contribution
posted on 2020-02-21, 21:44 authored by Biao Fu, Christian Brown, Lena MälerGalactolipids
are characteristic lipids of the photosynthetic membranes.
They are highly enriched in the chloroplast and are present in photosystem
structures. There are two major types of galactolipids, i.e., monogalactosyldiacylglycerol
and digalactosyldiacylglycerol (DGDG) in chloroplastic membranes,
which amount to ∼50 and ∼20 mol % of the total chloroplast
lipids, respectively. Under phosphate-limiting conditions, the amount
of DGDG increases dramatically for rescuing phosphate from phospholipids.
In Arabidopsis thaliana, the gene digalactosyldiacylglycerol
synthase 2 (DGD2) encodes a membrane-associated
glycosyltransferase. The gene expression is highly responsive to phosphate
starvation and is significantly upregulated in this case. To understand
the molecular mechanism of DGD2, we established a protocol for DGD2
expression and purification in an Escherichia coli-based system. The work involved optimization of the expression condition
and the purification protocol and a careful selection of buffer additives.
It was found that a removal of around 70 C-terminal residues was necessary
to produce a homogeneous monomeric protein sample with high purity,
which was highly active. The purified sample was characterized by
an activity assay for enzyme kinetics in which a range of membrane
mimetics with different lipid compositions were used. The results
demonstrate that DGD2 activity is stimulated by the presence of negatively
charged lipids, which highlight the importance of the membrane environment
in modulating the enzyme’s activity. The study also paves way
for future biophysical and structural studies of the enzyme.
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Keywords
enzyme kineticsphotosynthetic membranesmembrane-associated glycosyltransferaseChloroplast Outer Membrane-Associated GlycosyltransferaseGalactolipid Synthesis Galactolipidsmembrane mimeticsphotosystem structuresArabidopsis thaliana70 C-terminal residueschloroplastic membranesgene digalactosyldiacylglycerol synthase 2phosphate-limiting conditionschloroplast lipidsgene expressionDGDG increasesactivity assaymonomeric protein sampleDGD 2 expressionpurification protocolEscherichia coliphosphate starvationDGD 2lipid compositionsbuffer additivesexpression conditionDGD 2 activitymembrane environment
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