| In vitro CLE Peptide Bioactivity Assay on Plant Roots
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Author:
Date:
2015-12-20
[Abstract] Plant CLAVATA3/ESR (CLE)-related proteins play diverse roles in plant growth and development including regulating the development of root meristem. The mature functional forms of CLE peptides are typically 12-13 amino acids (aa) in length that are derived from the conserved C-termini of their precursor proteins. Genes encoding small secreted peptides sharing similarity to plant CLE proteins have recently been cloned from plant-parasitic nematodes, pests that infect many important crops. It is demonstrated that exogenous application of synthetic 12-14 aa CLE peptides corresponding to the CLE domain of their precursor proteins can suppress plant root growth. This protocol is to evaluate the bioactivity of CLE peptides originated from plant-parasitic nematodes by measuring the growth of ...
[摘要] 植物CLAVATA3/ESR（CLE）相关蛋白质在植物生长和发育中发挥多种作用，包括调节根分生组织的发育。 CLE肽的成熟功能形式通常为12-13个氨基酸（aa）长度，其衍生自其前体蛋白的保守C-末端。 最近从植物寄生线虫，感染许多重要作物的害虫克隆了编码与植物CLE蛋白相似的小分泌肽的基因。 证明外源应用对应于其前体蛋白的CLE结构域的合成12-14aa CLE肽可以抑制植物根生长。 该协议是通过测量CLE肽处理后植物根的生长或根尖分生组织（RAM）的大小来评价源自植物寄生线虫的CLE肽的生物活性。 用于研究的植物包括拟南芥和马铃薯。
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| Density Gradient Centrifugation for Enrichment and Identification of GFP-tagged Chitosomal Microvesicles of Filamentous Fungi
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Author:
Date:
2015-10-05
[Abstract] Density gradient centrifugation has been utilized to characterize the subcellular distribution of physiologically relevant enzymes in yeasts and filamentous fungi (Leal-Morales et al., 1988; Martínez et al., 1989; Kamada et al., 1991). This approach is now potentiated by protein tagging and live imaging techniques, which make possible to relate a single protein with, for example, a discrete population of intracellular vesicles and their in vivo dynamics (Verdín et al., 2009; Fajardo-Somera et al., 2013; Sánchez-León et al., 2015). Here, we describe the density gradient centrifugation and fractionation analysis of cell-free homogenates of a Neurospora crassa (N. crassa) strain that expresses CHS-6 chitin ...
[摘要] 已经使用密度梯度离心来表征生物相关酶在酵母和丝状真菌中的亚细胞分布（Leal-Morales等人，1988;Martínez等人，1989） ; Kamada等人，1991）。 这种方法现在通过蛋白质标记和实时成像技术加强，这使得可能将单个蛋白质与例如胞内囊泡的离散群体及其体内动力学联系起来（Verdín等人 al。，2009; Fajardo-Somera等人，2013;Sánchez-León等人，2015）。 在这里，我们描述了表达CHS-6几丁质合酶的粗糙脉孢菌（ N.crassa ）菌株的无细胞匀浆的密度梯度离心和分级分析 绿色荧光蛋白（Riquelme等人，2007）。
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| Expression and Partial Purification of His-tagged Proteins in a Plant System
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Author:
Date:
2015-09-05
[Abstract] Plant protein expression can be a challenging enterprise in any biochemical or molecular biology research project. Several heterologous systems like bacteria, yeast, insect cells and cell free systems have been used to produce plant proteins for in vitro experiments and structural characterization. However, due to particularities of plant proteins, for example the specific type and abundance of post-translational modifications (e.g. glycosylation), a plant system to express plant proteins is extremely desirable. The use of Nicotiana benthamiana (N. benthamiana) plants for protein expression has proven to be quick and reliable. To illustrate the robustness and rapidity of this system, recent efforts to produce the first protein based drug against the ...
[摘要] 植物蛋白表达可以是任何生物化学或分子生物学研究项目中的挑战性企业。几种异源系统如细菌，酵母，昆虫细胞和无细胞系统已被用于生产植物蛋白用于体外实验和结构表征。然而，由于植物蛋白的特殊性，例如翻译后修饰（例如糖基化）的特异性类型和丰度，表达植物蛋白的植物系统是非常需要的。已经证明使用本氏烟草（本生烟草）植物进行蛋白质表达是快速和可靠的。为了说明该系统的稳健性和快速性，最近在N中进行了生产针对埃博拉病毒的第一种基于蛋白质的药物的努力。本草案蛋白质表达系统（Choi等人，2015）。
该方案描述了一种简单的系统，用于表达和富集（亲和纯化） > N。本生烟草叶，其被成功地用于拟南芥果胶乙酰酯酶，PAE8和PAE9的表征中（de Souza等人，2014）。
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