Dissecting intracellular signaling pathways with membrane-permeable peptides.
Dissecting intracellular signaling pathways with membrane-permeable peptides.
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用膜渗透肽剖析细胞内信号传导途径。
DOI：
10.1126/stke.2000.47.pl1
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发表时间：
2000
期刊：
影响因子：
--
通讯作者：
Sanders-Bush,E
中科院分区：
文献类型：
--
作者：
Chang,MS;Tam,JP;Sanders-Bush,E
可以设计模拟蛋白质相互作用基序的肽，因此可以用于特异性和选择性地阻断先前已鉴定蛋白质相互作用的信号转导级联中的特定步骤。该协议描述了合成与膜渗透序列（MPS）偶联的肽的方法，该序列是根据卡波西成纤维细胞生长因子的信号序列设计的，之前已证明该序列可以跨细胞膜易位共价附着的货物肽。为了提高这些膜渗透肽的制备效率、产量和多功能性，设计了基于两个未受保护的肽片段的模块化合成策略。模块化合成策略允许分别合成MPS和功能肽。以这种方式，通过传统的氟烯基甲氧基-羰基(Fmoc)化学合成或衍生自重组表达的肽或蛋白质的功能结构域可以在商业上购买以加速合成。随后，可以使用一步缀合反应将 MPS 结构域连接至任何功能结构域。该协议提供了肽合成、MPS 激活以及后续缀合协议的详细方法。
Peptides can be designed that mimic protein interaction motifs and thus, can be used to specifically and selectively block particular steps in signal transduction cascades where protein interactions have been previously identified. This protocol describes methods to synthesize peptides coupled to a membrane-permeable sequence (MPS), designed from the signal sequence of Kaposi fibroblast growth factor, which has been previously shown to translocate covalently attached cargo peptides across the cell membrane. To increase efficiency, yield, and versatility in the preparation of these membrane-permeable peptides, a modular synthesis strategy based on two unprotected peptide segments was designed. The modular synthesis strategy allows the MPS and functional peptides to be synthesized separately. In this manner, the functional domain of a peptide or protein, synthesized by traditional fluoroenylmethyloxy-carbonyl (Fmoc) chemistry or derived from recombinant expression, may be purchased commercially to expedite synthesis. Subsequently, the MPS domain may be attached to any functional domain using a one-step conjugation reaction. This protocol provides detailed methods for peptide synthesis, activation of the MPS, and the subsequent conjugation protocol.