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Sci. STKE, 29 August 2000
Vol. 2000, Issue 47, p. pl1
[DOI: 10.1126/stke.2000.47.pl1]

PROTOCOLS

Dissecting Intracellular Signaling Pathways with Membrane-Permeable Peptides

Mike S. S. Chang1*, James P. Tam2, and Elaine Sanders-Bush1

1The Department of Pharmacology and Center for Molecular Neuroscience, Vanderbilt University School of Medicine, Nashville, TN 37232, USA. E-mail: Mike.Chang@vanderbilt.edu, Elaine.Bush@mcmail.vanderbilt.edu
2The Department of Microbiology and Immunology, Vanderbilt University School of Medicine, Nashville, TN 37232, USA. E-mail: Tamjp@ctrvax.vanderbilt.edu

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.

*Corresponding author, E-mail: Mike.Chang{at}vanderbilt.edu

Citation: M. S. S. Chang, J. P. Tam, E. Sanders-Bush, Dissecting Intracellular Signaling Pathways with Membrane-Permeable Peptides. Sci. STKE 2000, pl1 (2000).

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