Unintended perturbation of protein function using GFP nanobodies in human cells
Unintended perturbation of protein function using GFP nanobodies in human cells
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DOI：
10.1242/jcs.234955
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发表时间：
2019-11-01
影响因子：
4
通讯作者：
Royle, Stephen J.
中科院分区：
文献类型：
--
作者：
Kuey, Cansu;Larocque, Gabrielle;Royle, Stephen J.
使用基因组编辑用GFP标记感兴趣的蛋白质是研究细胞和发育生物学中蛋白质功能的流行方法。为了避免重新工程化细胞系或生物体以引入额外的标签，可以使用结合GFP的功能化纳米抗体来扩展GFP标签的功能。我们开发了功能化的纳米抗体，我们称之为“加密狗”，它可以将FKBP标签添加到GFP标记的目标蛋白质上，从而能够在GFP敲入细胞系中进行敲入实验。knocksideways的力量在于它允许研究人员快速将蛋白质从活性状态转换为非活性状态。我们表明，加密狗允许在基因组编辑的人类细胞中有效地敲除GFP标记的蛋白质。然而，我们发现与发动蛋白-2-GFP结合的纳米抗体在敲侧之前引起发动蛋白功能的抑制。GFP标记的肿瘤蛋白D54（TPD 54，也称为TPD 52 L2）在顺行运输中的功能也受到软件狗的干扰。虽然这些问题可能会限制加密狗的应用，但我们讨论了将其部署为细胞生物学工具的策略。本文对论文的第一作者进行了相关的第一人称采访。
Tagging a protein of interest with GFP using genome editing is a popular approach to study protein function in cell and developmental biology. To avoid re-engineering cell lines or organisms in order to introduce additional tags, functionalized nanobodies that bind GFP can be used to extend the functionality of the GFP tag. We developed functionalized nanobodies, which we termed 'dongles', that could add, for example, an FKBP tag to a GFP-tagged protein of interest, enabling knocksideways experiments in GFP knock-in cell lines. The power of knocksideways is that it allows investigators to rapidly switch the protein from an active to an inactive state. We show that dongles allow for effective knocksideways of GFP-tagged proteins in genome-edited human cells. However, we discovered that nanobody binding to dynamin-2-GFP caused inhibition of dynamin function prior to knocksideways. The function of GFP-tagged tumor protein D54 (TPD54, also known as TPD52L2) in anterograde traffic was also perturbed by dongles. While these issues potentially limit the application of dongles, we discuss strategies for their deployment as cell biological tools.This article has an associated First Person interview with the first author of the paper.