Polymeric microspheres as protein transduction reagents.
Polymeric microspheres as protein transduction reagents.
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DOI：
10.1074/mcp.o113.034900
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发表时间：
2014-06
期刊：
影响因子：
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通讯作者：
Hine AV
中科院分区：
文献类型：
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作者：
Nagel D;Behrendt JM;Chimonides GF;Torr EE;Devitt A;Sutherland AJ;Hine AV
发现一种未知蛋白质的功能，特别是一种既没有结构也没有功能相关性的蛋白质，是一项艰巨的任务。相互作用分析确定结合伴侣，而DNA转染，无论是瞬时的或稳定的，导致细胞内的表达，但不一定在生理相关的水平。理论上，直接细胞内蛋白质递送（蛋白质转导）提供了一种概念上更简单的替代方案，但在实践中，该方法存在问题。结构域如HIV达特蛋白是有价值的，但它们的有效性是蛋白质特异性的。类似地，通过内吞途径（例如使用脂质体）递送完整蛋白对于功能分析是有问题的，因为蛋白在内体/溶酶体中降解的可能性。因此，最近的报道表明，微球可以通过非内吞的，能量不依赖的途径将生物货物递送到细胞中，为功能蛋白的体外递送提供了令人兴奋和有前途的替代方案。为了充分利用这种前景，需要微球（i）稳定地连接到蛋白质，（ii）可以以良好的效率递送那些蛋白质，（iii）一旦进入细胞内就释放功能性蛋白质，以及（iv）允许伴随的追踪。在这里，我们报告的应用微球成功地解决所有这些标准同时，第一次。细胞摄取后，蛋白质释放由还原性细胞质环境自动催化。在细胞外，共价微球-蛋白质连接在37 °C下稳定≥90 h。使用保守的估计方法，74.3% ± 5.6%的细胞显示在孵育24小时后摄取这些微球，整个递送过程和细胞内蛋白释放发生在36小时内。该方法旨在用于体外功能性蛋白质研究，将使得能够在生理学相关水平上研究蛋白质递送的后果，而无需求助于核酸，并提供了商业蛋白质转染试剂如Chariot™的有用替代品。我们还提供了明确的免疫染色证据，以解决围绕基于FACS的微球摄取评估的剩余争议。
Discovering the function of an unknown protein, particularly one with neither structural nor functional correlates, is a daunting task. Interaction analyses determine binding partners, whereas DNA transfection, either transient or stable, leads to intracellular expression, though not necessarily at physiologically relevant levels. In theory, direct intracellular protein delivery (protein transduction) provides a conceptually simpler alternative, but in practice the approach is problematic. Domains such as HIV TAT protein are valuable, but their effectiveness is protein specific. Similarly, the delivery of intact proteins via endocytic pathways (e.g. using liposomes) is problematic for functional analysis because of the potential for protein degradation in the endosomes/lysosomes. Consequently, recent reports that microspheres can deliver bio-cargoes into cells via a non-endocytic, energy-independent pathway offer an exciting and promising alternative for in vitro delivery of functional protein. In order for such promise to be fully exploited, microspheres are required that (i) are stably linked to proteins, (ii) can deliver those proteins with good efficiency, (iii) release functional protein once inside the cells, and (iv) permit concomitant tracking. Herein, we report the application of microspheres to successfully address all of these criteria simultaneously, for the first time. After cellular uptake, protein release was autocatalyzed by the reducing cytoplasmic environment. Outside of cells, the covalent microsphere–protein linkage was stable for ≥90 h at 37 °C. Using conservative methods of estimation, 74.3% ± 5.6% of cells were shown to take up these microspheres after 24 h of incubation, with the whole process of delivery and intracellular protein release occurring within 36 h. Intended for in vitro functional protein research, this approach will enable study of the consequences of protein delivery at physiologically relevant levels, without recourse to nucleic acids, and offers a useful alternative to commercial protein transfection reagents such as Chariot™. We also provide clear immunostaining evidence to resolve residual controversy surrounding FACS-based assessment of microsphere uptake.