Cabantous Stéphanie, Terwilliger Thomas C, Waldo Geoffrey S
Bioscience Division, MS-M888, Los Alamos National Laboratory, PO Box 1663, Los Alamos, New Mexico 87545, USA.
Nat Biotechnol. 2005 Jan;23(1):102-7. doi: 10.1038/nbt1044. Epub 2004 Dec 5.
Existing protein tagging and detection methods are powerful but have drawbacks. Split protein tags can perturb protein solubility or may not work in living cells. Green fluorescent protein (GFP) fusions can misfold or exhibit altered processing. Fluorogenic biarsenical FLaSH or ReASH substrates overcome many of these limitations but require a polycysteine tag motif, a reducing environment and cell transfection or permeabilization. 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, but the GFP fragments reported thus far are large and fold poorly, require chemical ligation or fused interacting partners to force their association, or require coexpression or co-refolding to produce detectable folded and fluorescent GFP. 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.
现有的蛋白质标记和检测方法功能强大，但也存在缺点。分裂蛋白质标签可能会干扰蛋白质的溶解度，或者在活细胞中不起作用。绿色荧光蛋白（GFP）融合蛋白可能会错误折叠或表现出加工过程的改变。荧光双砷酸盐FLaSH或ReASH底物克服了这些局限性中的许多问题，但需要一个多聚半胱氨酸标签基序、一个还原环境以及细胞转染或通透处理。理想的蛋白质标签应该是基因编码的，能在体内和体外发挥作用，能提供灵敏的分析信号，并且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GFP，但迄今为止报道的GFP片段很大且折叠性差，需要化学连接或融合相互作用的伙伴来促使它们结合，或者需要共表达或共重折叠以产生可检测的折叠且有荧光的GFP。我们已经设计出了可溶的、能自我结合的GFP片段，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。分裂GFP系统简单，且不会改变融合蛋白的溶解度。
