| Isolation of the Dot/Icm Type IV Secretion System Core Complex from Legionella pneumophila for Negative Stain Electron Microscopy Studies
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Author:
Date:
2017-04-20
[Abstract] Legionella possesses a pivotal secretion machinery to deliver virulence factors to eukaryotic host cells. In this protocol, we describe the procedure for isolation of the native core complex of the Dot/Icm type IV secretion system from L. pneumophila aiming to perform biochemical and transmission electron microscopy analyses.
[摘要] 军团菌具有关键的分泌机制，以将毒力因子递送至真核宿主细胞。在本协议中，我们描述了从L / L分离Dot / Icm IV型分泌系统的天然核心复合物的步骤。肺炎支原体旨在进行生物化学和透射电子显微镜分析。
嗜肺军团菌是革兰氏阴性细菌病原体，其导致被称为退伍军人病的肺部感染（Fields等人，2002）。 L。嗜肺杆菌利用由 dot / cm 基因编码的IV型分泌系统（T4SS）将大约300种细菌蛋白转运到其真核宿主细胞质中以劫持细胞过程（Hubber和Roy， 2010）。由超过20种蛋白组成，T4SS是建立在细菌内膜和外膜上的纳米机器（Nagai和Kubori 2011; Kubori和Nagai 2016）。 Dot / Icm T4SS的核心复合物是系统的生物化学稳定部分，并形成桥接内膜和外膜的输送导管（Kubori等人，2014）。核心复合物由至少五种蛋白质组成;三种外膜相关蛋白，DotC，DotD和DotH，以及两种内膜蛋白DotF和DotG（Vincent等人，2006）。基于来自鼠伤寒沙门氏菌的另一种细菌纳米机械的III型分泌系统的生物化学分离方法（Kubori等人，1998; Marlovits等人， ...
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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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