| Expression and Purification of Mini G Proteins from Escherichia coli
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Author:
Date:
2017-04-20
[Abstract] Heterotrimeric G proteins modulate intracellular signalling by transducing information from cell surface G protein-coupled receptors (GPCRs) to cytoplasmic effector proteins. Structural and functional characterisation of GPCR–G protein complexes is important to fully decipher the mechanism of signal transduction. However, native G proteins are unstable and conformationally dynamic when coupled to a receptor. We therefore developed an engineered minimal G protein, mini-Gs, which formed a stable complex with GPCRs, and facilitated the crystallisation and structure determination of the human adenosine A2A receptor (A2AR) in its active conformation. Mini G proteins are potentially useful tools in a variety of applications, including characterising GPCR ...
[摘要] 异源三聚体G蛋白通过将细胞表面G蛋白偶联受体（GPCR）的信息转导至细胞质效应蛋白来调节细胞内信号传导。 GPCR-G蛋白复合物的结构和功能表征对于完全破译信号转导机制是重要的。然而，当与受体偶联时，天然G蛋白质是不稳定的并具有构象的动力学。因此，我们开发了一种工程化的最小G蛋白，其与GPCR形成稳定的复合物，促进了人腺苷A 2A受体的结晶和结构测定（A 2AR）的活性构象。 Mini G蛋白是各种应用中潜在有用的工具，包括表征GPCR药理学，结合亲和力和动力学实验，激动剂药物发现和GPCR-G蛋白复合物的结构测定。在这里，我们描述了一个用于表达和纯化mini-G 的详细方案。
我们最近报告了一种工程化的最小G蛋白质（Carpenter和Tate，2016）的开发，其促进了人腺苷A 2A受体的结晶（ A 2 R 2）其活性构象（Carpenter等人，2016; Carpenter和Tate，2017）。不同于需要在真核系统中表达的异源三聚体G蛋白质，在大肠杆菌（大肠杆菌）中高度表达微型G蛋白，并且可以可以容易地纯化，每升培养物的产量为50-100mg的mini-G 。在这里，我们描述了早先在Carpenter和Tate（2016）中描述的可以用于前面描述的任何一种迷你G蛋白构建体的表达和纯化的逐步方案（Carpenter等人， ...
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| Organotypic Spinal Cord Slice Cultures and a Method to Detect Cell Proliferation in These Slices
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Author:
Date:
2016-10-05
[Abstract] In these culture models, the normal cytoarchitecture and local neuronal circuits of the spinal cord are preserved, offering a compromise between dissociated cell cultures and complete animal studies. The addition of 5-ethynyl-2’-deoxyuridine (EdU) to the culture medium allows for the detection of proliferating cells.
[摘要] 在这些文化模型中，保留了脊髓的正常细胞结构和局部神经元回路，提供了解离的细胞培养物和完全动物研究之间的折衷。 向培养基中加入5-乙炔基-2'-脱氧尿苷（EdU）允许检测增殖细胞。
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| Dictyostelium Cultivation, Transfection, Microscopy and Fractionation
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Author:
Date:
2015-06-05
[Abstract] The real time visualisation of fluorescently tagged proteins in live cells using ever more sophisticated microscopes has greatly increased our understanding of the dynamics of key proteins during fundamental physiological processes such as cell locomotion, chemotaxis, cell division and membrane trafficking. In addition the fractionation of cells and isolation of organelles or known compartments can often verify any subcellular localisation and the use of tagged proteins as bait for the immunoprecipitation of material from cell fractions can identify specific binding partners and multiprotein complexes thereby helping assign a function to the tagged protein. We have successfully applied these techniques to the Dictyostelium discoideum protein TSPOON that is part of an ancient ...
[摘要] 使用更复杂的显微镜，活细胞中荧光标记的蛋白质的实时可视化大大增加了我们对基本生理过程如细胞运动，趋化性，细胞分裂和膜运输过程中关键蛋白质动力学的了解。此外，细胞的分级和分离细胞器或已知的隔室通常可以验证任何亚细胞定位，并且使用标记的蛋白质作为诱饵用于来自细胞部分的物质的免疫沉淀可以鉴定特异性结合配偶体和多蛋白复合物，从而有助于赋予功能标记蛋白。我们已经成功地将这些技术应用于作为古代异构六聚体膜转运复合物的一部分的盘基网柄菌discoideum蛋白TSPOON（Hirst等，2013）。 ...
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