Electroporated recombinant proteins as tools for in vivo functional complementation, imaging and chemical biology
Electroporated recombinant proteins as tools for in vivo functional complementation, imaging and chemical biology
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DOI：
10.7554/elife.48287
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发表时间：
2019-07-16
期刊：
影响因子：
7.7
通讯作者：
Maffini, Stefano
中科院分区：
文献类型：
--
作者：
Alex, Amal;Piano, Valentina;Maffini, Stefano
将天然或化学修饰的重组蛋白送入哺乳动物细胞显示出在功能研究和各种技术应用方面的前景，但人们对所传递蛋白的亚细胞定位和功能完整性可能受到影响的担忧仍然很高。在这里，我们考察了批量电穿孔作为一种运送工具的单一多肽和多亚单位蛋白组合的动粒，空间受限和研究良好的亚细胞结构。重组荧光Ndc80和Mis12多亚基复合体在电穿孔进入人细胞后，表现出天然定位、与内源性结合伙伴物理相互作用和功能互补的功能，从而促进有丝分裂检查点信号和染色体分离。Dynein接头的动粒定位需要法尼化。在长期抑制法尼基转移酶活性的细胞中，体外法尼化和重组Spinly的电穿孔确实导致了强大的动粒定位。我们的数据表明，电穿孔非常适合提供合成的和化学修饰的功能蛋白质版本，因此，构成了一种在化学和合成生物学中应用的有前途的工具。
Delivery of native or chemically modified recombinant proteins into mammalian cells shows promise for functional investigations and various technological applications, but concerns that sub-cellular localization and functional integrity of delivered proteins may be affected remain high. Here, we surveyed batch electroporation as a delivery tool for single polypeptides and multi-subunit protein assemblies of the kinetochore, a spatially confined and well-studied subcellular structure. After electroporation into human cells, recombinant fluorescent Ndc80 and Mis12 multi-subunit complexes exhibited native localization, physically interacted with endogenous binding partners, and functionally complemented depleted endogenous counterparts to promote mitotic checkpoint signaling and chromosome segregation. Farnesylation is required for kinetochore localization of the Dynein adaptor Spindly. In cells with chronically inhibited farnesyl transferase activity, in vitro farnesylation and electroporation of recombinant Spindly faithfully resulted in robust kinetochore localization. Our data show that electroporation is well-suited to deliver synthetic and chemically modified versions of functional proteins, and, therefore, constitutes a promising tool for applications in chemical and synthetic biology.