| Heterologous Expression and Purification of the CRISPR-Cas12a/Cpf1 Protein
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Author:
Date:
2018-05-05
[Abstract] This protocol provides step by step instructions (Figure 1) for heterologous expression of Francisella novicida Cas12a (previously known as Cpf1) in Escherichia coli. It additionally includes a protocol for high-purity purification and briefly describes how activity assays can be performed. These protocols can also be used for purification of other Cas12a homologs and the purified proteins can be used for subsequent genome editing experiments.
Figure 1. Timeline of activities for the heterologous expression and purification of Francisella novicida Cas12a (FnCas12a) from Escherichia coli
[摘要] 该协议提供了分步说明（图1），用于在大肠杆菌中异源表达新西兰弗朗西斯菌弗朗西丝菌Cas12a（以前称为Cpf1）。 它还包括一个高纯度纯化方案，并简要介绍如何进行活性测定。 这些方案也可以用于其他Cas12a同系物的纯化，并且纯化的蛋白质可以用于随后的基因组编辑实验。
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图1.从大肠杆菌 异源表达和纯化<弗朗西斯弗朗西丝菌 Cas12a（FnCas12a）的活动时间表
【背景】原核CRISPR-Cas免疫系统通过使用CRISPR RNA（crRNA）作为外源DNA或RNA的序列特异性靶向的指导来提供针对病毒和质粒的保护（van der Oost等人，2014; Marraffini ，2015）。 1类CRISPR-Cas系统（包含I型，III型和IV型）通常形成多亚基蛋白-cRNA效应复合物，而2类系统（包含II型，V型和VI型）依赖于单个crRNA-引导的效应物核酸酶用于目标干扰（Mohanraju et al。 2016年）。
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| Analysis of Phagosomal Antigen Degradation by Flow Organellocytometry
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Author:
Date:
2016-11-20
[Abstract] Professional phagocytes internalize self and non-self particles by phagocytosis to initiate innate immune responses. After internalization, the formed phagosome matures through fusion and fission events with endosomes and lysosomes to obtain a more acidic, oxidative and hydrolytic environment for the degradation of its cargo. Interestingly, phagosome maturation kinetics differ between cell types and cell activation states. This protocol allows to quantify phagosome maturation kinetics on a single organelle level in different types of phagocytes using flow cytometry. Here, ovalbumin (OVA)-coupled particles are used as phagocytosis model system in dendritic cells (DC), which are internalized by phagocytosis. After different time points, phagosome maturation parameters, such as phagosomal ...
[摘要] 专业吞噬细胞通过吞噬作用内化自身和非自身颗粒以启动先天免疫应答。内化后，形成的吞噬体通过与内体和溶酶体的融合和裂变事件成熟，以获得更酸性，氧化和水解的环境用于其货物的降解。有趣的是，吞噬体成熟动力学在细胞类型和细胞活化状态之间不同。该协议允许使用流式细胞术定量不同类型的吞噬细胞中单个细胞器水平上的吞噬体成熟动力学。在这里，卵白蛋白（OVA）耦合的颗粒用作吞噬作用模型系统在树突状细胞（DC），其通过吞噬内化。在不同的时间点之后，吞噬体成熟参数，例如OVA的吞噬体降解和溶酶体蛋白（例如LAMP-1）的获得，可以通过流式细胞器细胞计数以高度定量的方式同时测量。这些读出可以与其他吞噬体功能相关，例如抗原降解，在DC中的加工和负载。
[背景] ...
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| Spectrophotometric Determination of Glutamine Synthetase Activity in Cultured Cells
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Author:
Date:
2016-10-05
[Abstract] Glutamine synthetase (GS), which catalyzes the conversion of glutamate and ammonia to glutamine, is widely distributed in animal tissues and cell culture lines. The importance of this enzyme is suggested by the fact that glutamine, the product of GS-catalyzed de novo synthesis reaction, is the most abundant free amino acid in blood (Smith and Wilmore, 1990). Glutamine is involved in many biological processes including serving as the nitrogen donor for biosynthesis, as an exchanger for the import of essential amino acids, as a means to detoxifying intracellular ammonia and glutamate, and as a bioenergetics nutrient to fuel the tricarboxylic acid (TCA) cycle (Bott et al., 2015). The method for the assay of GS enzymatic activity relies on its γ-glutamyl transferase reaction by ...
[摘要] 谷氨酰胺合成酶（GS），其催化谷氨酸和氨转化成谷氨酰胺，广泛分布在动物组织和细胞培养系中。该酶的重要性通过谷氨酰胺，GS-催化的从头合成反应的产物，是血液中最丰富的游离氨基酸的事实提示（Smith和Wilmore，1990）。谷氨酰胺参与许多生物过程，包括作为生物合成的氮供体，作为输入必需氨基酸的交换剂，作为解毒细胞内氨和谷氨酸的手段，以及作为生物能量营养物来给三羧酸（TCA）周期（Bott等人，2015）。用于测定GS酶活性的方法依赖于其γ-谷氨酰转移酶反应，通过测量由谷氨酰胺和羟胺合成的γ-谷氨酰羟肟酸酯，以及反应产物与反应物的色谱分离（Deuel等人 。，1978）。 GS谷氨酰转移酶反应的概述可以在图1中找到。通过分光光度测定法在560nm的特定波长下使用酶标仪测量GS活性。该方法简单，并且具有与应用放射性标记的底物的那些方法相当的灵敏度。该修改的方法已经应用于在包括人乳腺上皮MCF10A细胞和鼠前B FL5.12细胞的培养细胞系中测定/测定GS活性，并且可以用于测量其他细胞系中的GS活性。 >
图1 。GS glutamyl ...
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