| Expression, Purification and Crystallization of Recombinant Arabidopsis Monogalactosyldiacylglycerol Synthase (MGD1)
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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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| Single Cell Flow Cytometry Assay for Peptide Uptake by Bacteria
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Author:
Date:
2016-12-05
[Abstract] Antimicrobial peptides (AMPs) can target the bacterial envelope or alternatively have intracellular targets. The latter requires uptake of the peptide by the bacterial cells. The bacterial internalization of an AMP can be evaluated by a fluorescence-based method that couples the use of the fluorescently labelled AMP to the fluorescence quencher trypan blue. Trypan blue is excluded from the interior of intact cells and the fluorescence of the extracellular peptide or of the peptide bound on the bacterial surface can be quenched by it, while the fluorescence of the internalized peptide is not affected. The uptake of the peptide by the bacteria is determined by measuring the fluorescence in individual cells by flow cytometry.
[摘要] 抗微生物肽（AMP）可靶向细菌包膜或者具有细胞内靶标。后者需要细菌细胞摄取肽。 AMP的细菌内化可以通过基于荧光的方法来评估，所述方法将荧光标记的AMP的使用耦合到荧光猝灭剂台盼蓝。台盼蓝从完整细胞的内部排除，并且细胞外肽或结合在细菌表面上的肽的荧光可被其淬灭，而内化肽的荧光不受影响。通过用流式细胞术测量单个细胞中的荧光来测定细菌对肽的吸收。
关键词：抗微生物肽，流式细胞术，肽摄取，肽转运蛋白，台盼蓝，碘化物吸收量
[背景] AMP由广泛且多样的有效抗微生物剂组成，具有作为新型治疗剂的潜力（Wang等人）。 ，2015）。 AMP是天然免疫的一部分并且由所有王国的生物产生。它们被这些生物体动员以抵抗感染的微生物，其可以是细菌，真菌或病毒。他们这样做通过直接杀死微生物，但他们也可以作为哨兵，促进其他免疫途径。有趣的是，还已经清楚的是，AMP不仅是针对不良微生物的药剂，而且它们还在动物和植物宿主中共生细菌群体的控制中具有关键作用（Maróti等人，2011 ; Kondorosi et al 。，2013）。
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