A molecular genetic approach to stabilizing bioactive peptides via protein-based motifs
Warren, James Walter
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Instability of bioactive peptides represents a major challenge to the development of these molecules as drugs. The purpose of this study was the investigation of protein-based motifs and their potential for protecting peptides from enzymatic degradation. A novel, highly regulable expression vector, pLAC11, was constructed so that potent inhibitory peptides could be isolated from an in vivo genetic screen designed to generate randomized bioactive peptides that inhibit the growth of bacteria. Compared to other commonly utilized expression vectors that are known not to be tightly regulable, pLAC11 was demonstrated to possess the ability to regulate potent inhibitor peptides which could prove lethal to the cells generating them. The three protein-based motifs that were examined by this screen were the fusion of randomized peptides to the highly stable Rop protein, the generation of !-helical peptides via the random incorporation of a set of hydrophilic helix-forming amino acids, and the use of oppositely charged residues at the termini of randomized peptides. All three methods were observed to yield an increase in the frequency at which potent inhibitors were isolated during the in vivo screen as compared to unprotected peptides, presumably due to greater stability. To further investigate the possible role of secondary structure in peptide stability, a group of !-helical peptides based on Rop and other helical proteins as well as putative helical peptides designed de novo were examined by secondary structure prediction algorithms, CD spectroscopy, and in vitro plasma degradation assays. Two highly stable helical peptides were identified and found to have half-lives much greater than other representative peptides and similar to that of small stable proteins.