Department of Chemistry and Pharmacy, Institute of Biochemistry, University of Münster, Corrensstrasse 36, 48149, Münster, Germany.
Department of Biology and Center for Cellular Nanoanalytics, University of Osnabrück, Barbarastrasse 11, 49076, Osnabrück, Germany.
Angew Chem Int Ed Engl. 2020 Nov 16;59(47):21007-21015. doi: 10.1002/anie.202006822. Epub 2020 Sep 11.
Protein semi-synthesis inside live cells from exogenous and endogenous parts offers unique possibilities for studying proteins in their native context. Split-intein-mediated protein trans-splicing is predestined for such endeavors and has seen some successes, but a much larger variety of established split inteins and associated protocols is urgently needed. We characterized the association and splicing parameters of the Gp41-1 split intein, which favorably revealed a nanomolar affinity between the intein fragments combined with the exceptionally fast splicing rate. Following bead-loading of a chemically modified intein fragment precursor into live mammalian cells, we fluorescently labeled target proteins on their N- and C-termini with short peptide tags, thus ensuring minimal perturbation of their structure and function. In combination with a nuclear-entrapment strategy to minimize cytosolic fluorescence background, we applied our technique for super-resolution imaging and single-particle tracking of the outer mitochondrial protein Tom20 in HeLa cells.
从外源和内源部分在活细胞内进行蛋白质半合成，为在天然环境中研究蛋白质提供了独特的可能性。分裂内含肽介导的蛋白质反式剪接非常适合于此类研究，并已取得了一些成功，但更需要更多种类的已建立的分裂内含肽和相关协议。我们对 Gp41-1 分裂内含肽的结合和剪接参数进行了表征，该内含肽有利地揭示了两个内含肽片段之间具有纳摩尔亲和力，同时具有异常快速的剪接速率。在将化学修饰的内含肽片段前体加载到活哺乳动物细胞后，我们用短肽标签对其 N-和 C-末端的靶蛋白进行荧光标记，从而确保对其结构和功能的最小干扰。结合核捕获策略以最小化细胞质荧光背景，我们将我们的技术应用于 HeLa 细胞中外线粒体蛋白 Tom20 的超分辨率成像和单颗粒跟踪。
