Chromophore-Assisted Laser Inactivation of α- and γ-Tubulin SNAP-tag Fusion Proteins inside Living Cells
Chromophore-Assisted Laser Inactivation of α- and γ-Tubulin SNAP-tag Fusion Proteins inside Living Cells
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DOI：
10.1021/cb800298u
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发表时间：
2009-02-01
影响因子：
4
通讯作者：
Ellenberg, Jan
中科院分区：
文献类型：
--
作者：
Keppler, Antje;Ellenberg, Jan
发色团辅助激光灭活（卡利）可以帮助解开活细胞内特定时间和位置的靶蛋白的局部活性。用荧光团如荧光素对融合蛋白进行共价SNAP标签标记是一种快速且高度特殊的工具，用于将光敏剂在体内附着到其靶蛋白上，以选择性灭活融合蛋白。在这里，我们通过在细胞分裂的活体成像测定期间α-微管蛋白和γ-微管蛋白SNAP-标签融合的急性失活来证明基于SNAP-标签的卡利的有效性和特异性。单线态氧被确认为导致融合蛋白功能丧失的活性氧物质。SNAP-tag卡利的主要优点是标记容易、可靠和高度灵活：基因编码的蛋白质标签可以用符合实验要求的各种染料共价标记。这使得SNAP标记卡利成为活细胞中标记蛋白质快速失活的非常有用的工具。
Chromophore-assisted laser inactivation (CALI) can help to unravel localized activities of target proteins at defined times and locations within living cells. Covalent SNAP-tag labeling of fusion proteins with fluorophores such as fluorescein is a fast and highly special tool to attach the photosensitizer to its target protein in vivo for selective inactivation of the fusion protein. Here, we demonstrate the effectiveness and specificity of SNAP-tag based CALI by acute inactivation of alpha-tubulin and gamma-tubulin SNAP-tag fusions during live imaging assays of cell division. Singlet oxygen is confirmed as the reactive oxygen species that leads to loss of fusion protein function. The major advantage of SNAP-tag CALI is the ease, reliability, and high flexibility labeling: the genetically encoded protein tag can be covalently labeled with various dyes matching the experimental requirements. This makes SNAP-tag CALI a very useful tool for rapid inactivation of tagged proteins in living cells.