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Author:
Date:
2016-12-20
[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.
[摘要] 在植物细胞中，半乳糖脂是主要的，代表高达光合组织脂质含量的50％。通过使用UDP-半乳糖作为供体糖和二酰基甘油（DAG）作为受体的MGDG合成酶（MGD）启动半乳糖脂合成以形成单糖基二酰基甘油（MGDG）。该方案用于在大肠杆菌中产生拟南芥（Arabidopsis thaliana）（拟南芥）单糖半乳糖二酰基甘油合酶1（MGD1）蛋白的重组形式（大肠杆菌），使用两步色谱纯化方法。蛋白质容易表达和纯化至数量，适用于生物化学和结构研究。还描述了MGD1的结晶。
背景 以前在e中表达植物MGD的尝试。大肠杆菌显示约99％的重组蛋白质积累在包涵体中（Miège等，1999）。开发了使用洗涤剂或体外包涵体折叠方案的细菌膜的溶解，并产生足够的纯和活性级分，足以监测酶的活性，但不进行其结构研究（Nishiyama et ...
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