Galactolipids 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.

中文翻译：

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DGD2的表达和纯化，DGD2是一种用于半乳糖脂合成的叶绿体外膜相关糖基转移酶。

半乳糖脂是光合膜的特征脂质。它们富含叶绿体，并存在于光系统结构中。半塑脂有两种主要类型，即叶绿塑料膜中的单半乳糖基二酰基甘油和二半乳糖基二酰基甘油（DGDG），分别占总叶绿体脂质的〜50和〜20 mol％。在磷酸盐限制条件下，DGDG的量急剧增加，以从磷脂中拯救磷酸盐。在拟南芥中，基因二半乳糖基二酰基甘油合成酶2（DGD2）编码与膜相关的糖基转移酶。该基因表达对磷酸盐饥饿高度敏感，并且在这种情况下显着上调。要了解DGD2的分子机制，我们建立了在基于大肠杆菌的系统中DGD2表达和纯化的协议。这项工作涉及表达条件和纯化方案的优化以及缓冲液添加剂的仔细选择。已经发现，除去约70个C-末端残基对于生产具有高活性的高纯度的均质单体蛋白质样品是必要的。通过酶动力学活性测定来表征纯化的样品，其中使用了一系列具有不同脂质组成的膜模拟物。结果表明，带负电荷的脂质的存在刺激了DGD2的活性，这突出了膜环境在调节酶活性中的重要性。该研究还为酶的未来生物物理和结构研究铺平了道路。