| Investigating Localization of Chimeric Transporter Proteins within Chloroplasts of Arabidopsis thaliana
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Author:
Date:
2018-02-05
[Abstract] In this protocol, we describe a method to design chimeric proteins for specific targeting to the inner envelope membrane (IEM) of Arabidopsis chloroplasts and the confirmation of their localization by biochemical analysis. Specific targeting to the chloroplast IEM can be achieved by fusing the protein of interest with a transit peptide and an IEM targeting signal. This protocol makes it possible to investigate the localization of chimeric proteins in chloroplasts using a small number of transgenic plants by using a modified method of chloroplast isolation and fractionation. IEM localization of chimeric proteins can be further assessed by trypsin digestion and alkaline extraction. Here, the localization of the chimeric bicarbonate transporter, designated as SbtAII, is detected by ...
[摘要] 在这个协议中，我们描述了一种设计嵌合蛋白的方法，用于特异性靶向拟南芥叶绿体的内包膜（IEM）并通过生化分析确定它们的定位。 叶绿体IEM的特异性靶向可通过将感兴趣的蛋白质与转运肽和IEM靶向信号融合来实现。 这个协议使得有可能使用少量的转基因植物，通过使用修改的叶绿体分离和分离方法来研究嵌合蛋白在叶绿体中的定位。 嵌合蛋白的IEM定位可以通过胰蛋白酶消化和碱性提取进一步评估。 在此，称为SbtAII的嵌合碳酸氢根转运蛋白的定位通过使用针对葡萄球菌蛋白A的抗体进行蛋白质印迹来检测。该方案改编自上原等人，2016年
【背景】有人提出将蓝藻CO 2浓度机制整合到叶绿体中是改善C 3+植物光合作用的有希望的方法。 根据理论估计，将BicA和SbtA整合到叶绿体IEM中可以提高光合CO 2固定率。 我们研究了核编码的蓝细菌碳酸氢盐转运蛋白BicA和SbtA与拟南芥叶绿体的IEM的整合。 因此，我们制定了一个协议，设计嵌合构造为特定目标的IEM和调查嵌合蛋白在叶绿体中的定位。
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| Extraction and Measurement of Abscisic Acid in a Unicellular Red Alga Cyanidioschyzon merolae
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Author:
Date:
2016-12-05
[Abstract] Abscisic acid (ABA) has been known as a phytohormone of land plants, which is synthesized in response to abiotic stresses and induces various physiological responses, but is also found from eukaryotic algae. Recently, we reported that a unicellular red alga Cyanidioschyzon merolae produced ABA, which prevented cell growth and enhanced salt stress tolerance (Kobayashi et al., 2016). This report describes the detailed method for the extraction and quantification of ABA in the model red alga C. merolae.
[摘要] 脱落酸（ABA）已知为陆生植物的植物激素，其响应于非生物胁迫合成并诱导各种生理反应，但也可从真核藻类中发现。最近，我们报道了单细胞红藻（Cyanidioschyzon merolae）产生ABA，其阻止细胞生长和增强盐胁迫耐受性（Kobayashi等人，2016）。该报告描述了在红海藻模型中提取和定量ABA的详细方法。 。
关键字：脱落酸，藻类， Cyanidioschyzon merolae ，HPLC，植物激素
] 植物激素ABA已在发散光合真核生物中发现，但单细胞藻类的功能仍不清楚。在最近的研究中，我们显示单细胞红藻。 melorae 通过本方案累积ABA以应答盐胁迫。这是用于从C中提取和定量ABA的第一已公布方案的细节。 merolae 。此协议针对 C进行了优化。基于陆地植物协议的。
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| Purification of the GfsA-3x FLAG Protein Expressed in Aspergillus nidulans
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Author:
Date:
2014-09-05
[Abstract] GfsA is a fungal β-galactofuranosyltransferase involved in the biosynthesis of O-glycan. To investigate the enzymatic functions of GfsA, we attempted to obtain a recombinant protein of this enzyme from two heterologous host organisms. However, GfsA could not be expressed as a recombinant protein in either Escherichia coli (E. coli) or Saccharomyces cerevisiae (S. cerevisiae). Therefore, we decided to employ Aspergillus nidulans (A. nidulans) as the host organism, and produced a strain that expressed 3x FLAG-tagged GfsA using chromosomal tagging. To confirm its expression, a solubilized protein was prepared from the tagged strain and analyzed with an anti-FLAG antibody. The strain that expressed 3x FLAG-tagged GfsA produced a ...
[摘要] GfsA是涉及O - 聚糖的生物合成的真菌β-半乳糖呋喃糖基转移酶。 为了研究GfsA的酶功能，我们尝试从两种异源宿主生物体获得该酶的重组蛋白。 然而，GfsA不能在大肠杆菌（大肠杆菌）或酿酒酵母（Saccharomyces cerevisiae）中表达为重组蛋白质（ cerevisiae ）。 因此，我们决定使用构巢曲霉（ A。nidulans ）作为宿主生物体，并产生使用染色体标签表达3×FLAG标记的GfsA的菌株。 为了证实其表达，从标记的菌株制备溶解的蛋白并用抗FLAG抗体分析。 表达3×FLAG标记的GfsA的菌株产生质量为约67kDa的功能性蛋白质。 该手稿中描述的方法允许纯化在A中表达的GfsA-3xFLAG蛋白。 构巢细胞。
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