| 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 ...
[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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| Determination of Recombinant Mannitol-1-phosphate Dehydrogenase Activity from Ectocarpus sp.
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Author:
Date:
2016-11-05
[Abstract] Brown algae belong to a phylogenetic lineage distantly related to green plants and animals, and are found predominantly, but not exclusively, in the intertidal zone, a harsh and frequently changing environment. Because of their unique evolutionary history and of their habitat, brown algae feature several peculiarities in their metabolism. One of these is the mannitol cycle, which plays a central role in their physiology, as mannitol acts as carbon storage, osmoprotectant, and antioxidant. This polyol is derived directly from the photoassimilate fructose-6-phosphate via the action of a ...
[摘要] 褐藻属于与绿色植物和动物遥远相关的系统发生谱系，并且主要发现于但不限于潮间带，一种苛刻且频繁变化的环境。由于它们独特的进化史和它们的栖息地，褐藻具有其代谢中的一些特性。其中之一是甘露醇循环，其在其生理学中发挥中心作用，因为甘露糖醇充当碳储存，渗透保护剂和抗氧化剂。该多元醇通过甘露醇-1-磷酸酯脱氢酶（M1PDH，EC 1.1.1.17）和甘露醇-1-磷酸酶（M1Pase，EC 3.1.3.22）的作用直接从光生酸酯果糖-6-磷酸衍生。该协议描述了在Ectocarpus中鉴定的三种M1PDH之一的重组催化结构域的生物化学表征。该重组催化结构域（下文称为M1PDHcat）使用NAD（H）作为辅因子催化果糖-6-磷酸（F6P）向甘露醇-1-磷酸（M1P）的可逆转化。 M1PDHcat活性在两个方向上测定，即，F6P还原和M1P氧化（图1）。
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图1.甘露醇-1-磷酸脱氢酶的可逆反应
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| Isolation of Tonoplast Vesicles from Tomato Fruit Pericarp
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Author:
Date:
2015-12-20
[Abstract] This protocol describes the isolation of tonoplast vesicles from tomato fruit. The vesicles isolated using this procedure are of sufficiently high purity for downstream proteomic analysis whilst remaining transport competent for functional assays. The methodology was used to study the transport of amino acids during tomato fruit ripening (Snowden et al., 2015) and based on the procedure used by Betty and Smith (Bettey and Smith, 1993). Such vesicles may be useful in further studies into the dynamic transfer of metabolites across the tonoplast for storage and metabolism during tomato ...
[摘要] 这个协议描述了从番茄果实隔离tonoplast囊泡。 使用该程序分离的囊泡具有足够高的纯度用于下游蛋白质组分析，同时保留用于功能测定的转运感受态。 该方法用于研究番茄果实成熟期间氨基酸的转运（Snowden等人，2015）并且基于Betty和Smith使用的程序（Bettey和Smith，1993）。 这样的囊泡可用于进一步研究代谢物在整个质膜上的动态转移，用于番茄果实发育期间的储存和代谢。
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