| 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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| PNGase Sensitivity Assay to Study the Folding Status of Proteins
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Author:
Date:
2016-10-05
[Abstract] This protocol aims to evaluate folding status of proteins, utilizing peptide:N-glycanase (PNGase) sensitivity. In the cytosol, PNGase works as a deglycosylation-enzyme. N-glycans on unfolded/misfolded proteins are more susceptible to PNGase than N-glycans on folded proteins because of the preference of PNGase to non-native proteins. PNGase is endogenously expressed in various cell types, including HCT116 cells, DT40 cells and mouse embryonic fibroblast cells. Partial deglycosylation by PNGase can be detected by faster migration of band in SDS-PAGE. You can compare tightness of the folding among wild-type and mutant proteins of interest. This method can be used with regular molecular and cell biology equipment, but applied only to glycoproteins.
[摘要] 该协议旨在评估蛋白质的折叠状态，利用肽：N-聚糖酶（PNGase）灵敏度。 在细胞质中，PNGase作为去糖基化酶。 由于PNG酶对非天然蛋白的偏好，解折叠/错折叠蛋白上的N-聚糖比折叠蛋白上的N-聚糖更易受PNG酶的影响。 PNGase在多种细胞类型中内源表达，包括HCT116细胞，DT40细胞和小鼠胚胎成纤维细胞。 通过PNGase的部分去糖基化可以通过在SDS-PAGE中更快的条带迁移来检测。 您可以比较感兴趣的野生型和突变蛋白之间折叠的紧密度。 该方法可以与常规的分子和细胞生物学设备一起使用，但仅应用于糖蛋白。
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| Trypsin Sensitivity Assay to Study the Folding Status of Proteins
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Author:
Date:
2016-10-05
[Abstract] This protocol aims to evaluate folding status of proteins, utilizing trypsin sensitivity. Unfolded/misfolded proteins are more susceptible to trypsin than folded proteins, because trypsin easily accesses and cleaves loosely folded parts of proteins. This method is especially useful to compare tightness of the folding among wild-type and mutant proteins. As trypsin generally cleaves a peptide bond at the carboxyl-terminal side of the amino acids lysine or arginine, this method can be used to analyze the folding status of different types of proteins such as integral membrane or soluble proteins (Ninagawa et al., 2015) and is applicable to cell lysates of any species and tissues as well as to recombinant proteins. You can use this technique with regular molecular and cell biology ...
[摘要] 该协议旨在评估蛋白质的折叠状态，利用胰蛋白酶敏感性。 由于胰蛋白酶容易进入和切割松散折叠的蛋白质部分，展开的/错误折叠的蛋白质比折叠的蛋白质更易于胰蛋白酶。 这种方法特别适用于比较野生型和突变型蛋白质之间折叠的紧密度。 由于胰蛋白酶通常在氨基酸赖氨酸或精氨酸的羧基末端侧切割肽键，所以该方法可用于分析不同类型蛋白质如整合膜或可溶性蛋白质的折叠状态（Ninagawa等，2015 ），适用于任何物种和组织以及重组蛋白的细胞裂解物。 您可以使用这种技术与常规分子和细胞生物学设备。
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