Cell-penetrating penta-peptides and Bax-inhibiting peptides: protocol for their application

The first series of cell-penetrating penta-peptides (CPP5s) were discovered as cytoprotective penta-peptides designed from the Bax-inhibiting domain of Ku70. Bax is an inducer of programmed cell death, and Ku70 is a multifunctional protein maintaining genomic stability and protecting cells from death by inhibiting the cytotoxic activity of Bax. Since these peptides bind and inhibit Bax, they are named Bax-inhibiting peptides (BIPs). The second series of CPP5s were developed by mutating BIP's amino acid sequences to abolish the Bax-binding activity. These peptides were used as negative control peptides to evaluate the Bax-inhibiting activity of BIPs. CPP5s are able to enter cells when they are added to the culture medium. The mechanism of cell entry of CPP5s is not yet understood. Numerous studies showed that BIP rescued cells from cytotoxic stresses both in cell culture and animal model, suggesting the therapeutic potential of BIP. Both BIPs and noncytoprotective CPP5s did not show significant toxicity even at 1.6 mM concentration in cell culture. Our recent study suggests that CPP5s has the protein transduction activity, though only green fluorescent protein (GFP) has been tested as a cargo protein. If CPP5s can deliver wide range of cargo molecules into the cell, CPP5s may be utilized as nontoxic drug delivery tool. In this article, we describe our laboratory's protocols of how to synthesize, store, and apply CPP5s for the examination of their activities of cell penetration and cytoprotection.