Protocol

Dissecting Intracellular Signaling Pathways with Membrane-Permeable Peptides

Science's STKE  29 Aug 2000:
Vol. 2000, Issue 47, pp. pl1
DOI: 10.1126/stke.2000.47.pl1

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Abstract

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.

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