In the work presented here we have used a function-based approach to isolate 12 novel cell-penetrating peptides from a 10,000+ member peptide library of rational design. Our unique high-throughput screen differentiates non-membranolytic from membranolytic translocation of peptides across lipid bilayers, thus allowing the selection of potential cell-penetrating peptides over potential antimicrobial peptides or peptide toxins. The 12 residue framework of the peptide library, designed with translocation in mind, is a series of 9 combinatorial sites followed by a C-terminal α-1-chymotrypsin cleavage site that is integral to the screen. The resulting residue in each of the combinatorial sites is one of 2 - 4 variable amino acids, with a hydrophobic or cationic residue available in each position. The sequences of nonpore-forming translocating peptides pulled from the screen have a 3 residue motif, Leu-Leu-Arg (p=10-5), and an overall under representation of basic residues in favor of hydrophobic amino acids. Upon characterization, these novel peptides were shown to behave akin to known cell-penetrating peptides found in nature. Ex vivo studies, in mammalian tissue cultures, revealed that the peptides translocate across the cell membrane without toxicity to the cell. In addition, structural studies showed a lack of convergence regarding a structure- function relationship, a continued trend seen among membrane-active peptides. In the course of the screen and the ex vivo studies, the peptides carried small polar molecules across lipid bilayers and biological membranes respectively; suggesting that, in addition to being cell-penetrating peptides, they could be put to use as effective therapeutic agents. The discovery of these novel cell-penetrating peptides by use of our screen supports function-based screening of peptide libraries as the best way to arrive at de novo membrane-active peptides with specific functions of interest. / acase@tulane.edu
| Identifer | oai:union.ndltd.org:TULANE/oai:http://digitallibrary.tulane.edu/:tulane_27207 |
| Date | January 2013 |
| Contributors | Marks, Jessica R. (Author), Wimley, William (Thesis advisor) |
| Publisher | Tulane University |
| Source Sets | Tulane University |
| Language | English |
| Detected Language | English |
| Format | 161 |
| Rights | Copyright is in accordance with U.S. Copyright law |
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