Nanobody-Based Probes for Subcellular Protein Identification and Visualization.
Nanobody-Based Probes for Subcellular Protein Identification and Visualization.
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DOI：
10.3389/fncel.2020.573278
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发表时间：
2020
影响因子：
5.3
通讯作者：
Giepmans BNG
中科院分区：
文献类型：
--
作者：
de Beer MA;Giepmans BNG
蛋白质鉴定和细胞定位在很大程度上有助于理解生命的组成成分对生理学的贡献。过去几十年发展起来的两种主要标记方法是用免疫球蛋白G(IGGS)等抗体标记或使用荧光蛋白等遗传编码标记。然而，IGG是大的蛋白质(150 KDa)，这限制了穿透深度和由所选择的靶向方法产生的长达∼25 nm的标记距离而导致的靶点位置的不确定性。此外，由于IGGS由多个独立的翻译链组成，因此不能作为融合蛋白的一部分进行重组调节和工程设计。在过去的十年里，单域抗原结合蛋白作为揭示分子身份和定位的工具被生物科学所探索，以克服免疫球蛋白的局限性。与常规应用相比，这些纳米体有几个潜在的好处。由于其尺寸小(15 KDa)，纳米体在标记过程中更好地穿透并提高分辨率。此外，纳米体能很容易地与其他基因融合。因此，可以很容易地设计出多结构域蛋白质，这些结构域包括靶向(纳米体)和通过荧光显微镜(荧光蛋白)或电子显微镜(基于某些酶)显示的结构域。还可以容易地添加用于例如提纯的附加模块。这些基于纳米体的探针可以应用于细胞中，用于活细胞内源性蛋白的检测，或者可以在用于分子、细胞或组织之前进行纯化。在这里，我们介绍了基于纳米体的探针的现状及其在显微镜下的实现，包括陷阱和潜在的未来机会。
Understanding how building blocks of life contribute to physiology is greatly aided by protein identification and cellular localization. The two main labeling approaches developed over the past decades are labeling with antibodies such as immunoglobulin G (IgGs) or use of genetically encoded tags such as fluorescent proteins. However, IgGs are large proteins (150 kDa), which limits penetration depth and uncertainty of target position caused by up to ∼25 nm distance of the label created by the chosen targeting approach. Additionally, IgGs cannot be easily recombinantly modulated and engineered as part of fusion proteins because they consist of multiple independent translated chains. In the last decade single domain antigen binding proteins are being explored in bioscience as a tool in revealing molecular identity and localization to overcome limitations by IgGs. These nanobodies have several potential benefits over routine applications. Because of their small size (15 kDa), nanobodies better penetrate during labeling procedures and improve resolution. Moreover, nanobodies cDNA can easily be fused with other cDNA. Multidomain proteins can thus be easily engineered consisting of domains for targeting (nanobodies) and visualization by fluorescence microscopy (fluorescent proteins) or electron microscopy (based on certain enzymes). Additional modules for e.g., purification are also easily added. These nanobody-based probes can be applied in cells for live-cell endogenous protein detection or may be purified prior to use on molecules, cells or tissues. Here, we present the current state of nanobody-based probes and their implementation in microscopy, including pitfalls and potential future opportunities.
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影响因子：
3.7
作者：
Buchfellner, Andrea;Yurlova, Larisa;Rothbauer, Ulrich
通讯作者：
Rothbauer, Ulrich
影响因子：
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作者：
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通讯作者：
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影响因子：
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作者：
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通讯作者：
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影响因子：
7.3
作者：
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通讯作者：
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影响因子：
4.6
作者：
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通讯作者：
Rothbauer U