Comparative Application of BioID and TurboID for Protein-Proximity Biotinylation
Comparative Application of BioID and TurboID for Protein-Proximity Biotinylation
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DOI：
10.3390/cells9051070
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发表时间：
2020-05-01
期刊：
影响因子：
6
通讯作者：
Roux, Kyle J.
中科院分区：
文献类型：
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作者：
May, Danielle G.;Scott, Kelsey L.;Roux, Kyle J.
BioID是一种用于鉴定蛋白质-蛋白质相互作用的成熟方法，已在活细胞和几种动物模型中使用。然而，常规标记周期需要15-18小时用于稳健的生物素化，这对于某些应用可能不是理想的。最近，使用BioID连接酶的定向进化开发了两种称为TurboID和miniTurbo的新连接酶，并且在与过量生物素孵育10分钟后能够产生稳健的生物素化。然而，有报道关注生物素化的诱导、细胞毒性和连接酶稳定性。为了进一步研究TurboID的实际应用，并确定与BioID相比的优势和劣势，我们开发了几种表达BioID和TurboID融合蛋白的稳定细胞系，并通过免疫印迹，免疫荧光和基于生物素亲和纯化的蛋白质组学对其进行分析。对于TurboID，我们观察到蛋白质不稳定的迹象，在不存在外源生物素的情况下持续的生物素化，以及实际标记半径的增加。然而，与BioID相比，TurboID能够在内质网腔中实现稳健的生物素化。生物素化的诱导可以通过将强力霉素诱导的表达与在生物素耗尽的培养基中的生长相结合来实现。这些研究应有助于告知研究人员利用基于BioID的方法，以适当的连接酶和实验方案，为他们的特殊需要。
BioID is a well-established method for identifying protein-protein interactions and has been utilized within live cells and several animal models. However, the conventional labeling period requires 15-18 h for robust biotinylation which may not be ideal for some applications. Recently, two new ligases termed TurboID and miniTurbo were developed using directed evolution of the BioID ligase and were able to produce robust biotinylation following a 10 min incubation with excess biotin. However, there is reported concern about inducibility of biotinylation, cellular toxicity, and ligase stability. To further investigate the practical applications of TurboID and ascertain strengths and weaknesses compared to BioID, we developed several stable cell lines expressing BioID and TurboID fusion proteins and analyzed them via immunoblot, immunofluorescence, and biotin-affinity purification-based proteomics. For TurboID we observed signs of protein instability, persistent biotinylation in the absence of exogenous biotin, and an increase in the practical labeling radius. However, TurboID enabled robust biotinylation in the endoplasmic reticulum lumen compared to BioID. Induction of biotinylation could be achieved by combining doxycycline-inducible expression with growth in biotin depleted culture media. These studies should help inform investigators utilizing BioID-based methods as to the appropriate ligase and experimental protocol for their particular needs.