Protein tagging and detection with engineered self-assembling fragments of green fluorescent protein
Protein tagging and detection with engineered self-assembling fragments of green fluorescent protein
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DOI：
10.1038/nbt1044
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发表时间：
2005-01-01
影响因子：
46.9
通讯作者：
Waldo, GS
中科院分区：
文献类型：
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作者：
Cabantous, S;Terwilliger, TC;Waldo, GS
现有的蛋白质标记和检测方法是强大的，但有缺点。分裂的蛋白质标签可以扰乱蛋白质溶解度（1-4）或可能在活细胞中不起作用（5-7）。绿色荧光蛋白（GFP）融合体可以错误折叠（8）或表现出改变的加工（9）。荧光双砷FLaSH或ReASH（10）底物克服了许多这些限制，但需要多聚半胱氨酸标签基序，还原环境和细胞转染或透化（10）。理想的蛋白质标签将是遗传编码的，将在体内和体外都起作用，将提供灵敏的分析信号，并且将不需要外部化学试剂或底物。实现这一点的一种方法可能是使用分裂的GFP（11），但是迄今为止报道的GFP片段很大并且折叠较差（11，12），需要化学连接（13）或融合的相互作用配偶体以迫使它们缔合（11-14），或者需要共表达或共重折叠以产生可检测的折叠和荧光GFP（11，12）。我们已经设计了可溶性的自缔合GFP片段，其可用于标记和检测活细胞或细胞裂解物中的可溶性或不溶性蛋白质。裂解GFP系统简单，不改变融合蛋白的溶解度。
Existing protein tagging and detection methods are powerful but have drawbacks. Split protein tags can perturb protein solubility(1-4) or may not work in living cells(5-7). Green fluorescent protein (GFP) fusions can misfold(8) or exhibit altered processing(9). Fluorogenic biarsenical FLaSH or ReASH(10) substrates overcome many of these limitations but require a polycysteine tag motif, a reducing environment and cell transfection or permeabilization(10). An ideal protein tag would be genetically encoded, would work both in vivo and in vitro, would provide a sensitive analytical signal and would not require external chemical reagents or substrates. One way to accomplish this might be with a split GFP(11), but the GFP fragments reported thus far are large and fold poorly(11,12), require chemical ligation(13) or fused interacting partners to force their association(11-14), or require coexpression or co-refolding to produce detectable folded and fluorescent GFP(11,12). We have engineered soluble, self-associating fragments of GFP that can be used to tag and detect either soluble or insoluble proteins in living cells or cell lysates. The split GFP system is simple and does not change fusion protein solubility.