Split-Inteins for Simultaneous, site-specific conjugation of Quantum Dots to multiple protein targets In vivo
Split-Inteins for Simultaneous, site-specific conjugation of Quantum Dots to multiple protein targets In vivo
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DOI：
10.1186/1477-3155-9-37
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发表时间：
2011-09-15
影响因子：
10.2
通讯作者：
Skourides, Paris A.
中科院分区：
文献类型：
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作者：
Charalambous, Anna;Antoniades, Ioanna;Skourides, Paris A.
背景资料：用量子点（QD）标记的蛋白质可以在长时间内以高的空间和时间分辨率成像，产生关于活细胞或体内蛋白质时空动力学的重要信息。然而，关于将QD用于生物成像的主要问题之一是难以将QD靶向蛋白质。我们最近开发了一种基于DnaE分裂内含肽的方法，在体内将量子点（QD）偶联到靶蛋白的C-末端。在这项研究中，我们扩展这种方法来实现特定位点的量子点的共轭两个或更多的蛋白质同时与光谱可区分的量子点的多参数成像的cellularfunctions.Results：使用DnaE分裂内含肽，我们的目标量子点的C-末端的桩蛋白，并显示桩蛋白-量子点共轭物成为本地化的焦点粘连，使成像的形成和溶解这些复合物。我们继续利用不同的分裂内含肽，即Ssp DnaB迷你内含肽，以证明N-末端蛋白质标记的量子点。这两个内含肽系统的组合允许我们在体内同时靶向两种具有光谱可区分的QD的不同蛋白质，而两个内含肽系统之间没有任何串扰。多靶标记是基于内含肽的方法的独特特征，其将其与现有的标记方法区分开来，因为考虑到大量表征的分裂内含肽，可以同时标记的单个靶的数量仅受可以在细胞内光谱区分的QD的数量限制。因此，内含肽介导的方法，同时，在体内，位点特异性（N-和C-末端）共轭量子点的多个蛋白质的目标开辟了新的可能性，生物成像应用，并提供了一个有效的系统，目标量子点和其他纳米结构的细胞内室以及特定的分子复合物。
Background: Proteins labelled with Quantum Dots (QDs) can be imaged over long periods of time with ultrahigh spatial and temporal resolution, yielding important information on the spatiotemporal dynamics of proteins within live cells or in vivo. However one of the major problems regarding the use of QDs for biological imaging is the difficulty of targeting QDs onto proteins. We have recently developed a DnaE split intein-based method to conjugate Quantum Dots (QDs) to the C-terminus of target proteins in vivo. In this study, we expand this approach to achieve site-specific conjugation of QDs to two or more proteins simultaneously with spectrally distinguishable QDs for multiparameter imaging of cellular functions.Results: Using the DnaE split intein we target QDs to the C-terminus of paxillin and show that paxillin-QD conjugates become localized at focal adhesions allowing imaging of the formation and dissolution of these complexes. We go on to utilize a different split intein, namely Ssp DnaB mini-intein, to demonstrate N-terminal protein tagging with QDs. Combination of these two intein systems allowed us to simultaneously target two distinct proteins with spectrally distinguishable QDs, in vivo, without any cross talk between the two intein systems.Conclusions: Multiple target labeling is a unique feature of the intein based methodology which sets it apart from existing tagging methodologies in that, given the large number of characterized split inteins, the number of individual targets that can be simultaneously tagged is only limited by the number of QDs that can be spectrally distinguished within the cell. Therefore, the intein-mediated approach for simultaneous, in vivo, site-specific (N- and C-terminus) conjugation of Quantum Dots to multiple protein targets opens up new possibilities for bioimaging applications and offers an effective system to target QDs and other nanostructures to intracellular compartments as well as specific molecular complexes.