Using protein‐based motifs to stabilize peptides

Abstract

While the use of synthetically derived novel inhibitor peptides as a source of new therapeutics for medicine remains incredibly promising, there is a major problem with implementing this technology, as many synthetic peptides have proven to be unstable and are degraded by peptidases in the host cell. In this study, we have investigated methods by which peptides can be stabilized using protein-based motifs in order to prevent the action of peptidases. Using an in vivo approach our laboratory developed to screen for synthetic peptides which can inhibit the growth of Escherichia coli, we found that protecting the amino or carboxyl terminus of the peptides via fusion to the very stable Rop protein, or the incorporation of two proline residues, increased the frequency at which potent inhibitor peptides could be isolated. Using an in vitro degradation assay in which extracts from several different cell types were tested, we demonstrated that peptides stabilized with multiple proline residues were more resistant to degradation than peptides stabilized by amidation or acetylation, two approaches that are routinely utilized to improve the stability of peptide drugs.

Construction and analysis of variants of a polyvalent Lyme disease vaccine: Approaches for improving the immune response to chimeric vaccinogens

“…In that case, it was speculated that structural issues or proteolytic degradation by carboxypeptidases was responsible [39][40][41]. Numerous methods have been proposed for protection of peptides and recombinant proteins from protease activity, including amidation or PEGylation of the C-terminus, acetylation of the amino (N)-terminus, and addition of protective amino acid motifs [42][43][44][45][46][47]. Two protective motifs, Gly-Gly and Pro-Pro-Ala, were assessed for their ability to enhance antibody responses to the ABKD vaccinogen.…”

“…The addition of neutral, hydrophilic Gly residues may reduce the activity of carboxypeptidases C and D, which are specific for hydrophobic and basic C-terminal amino acids, respectively [45,46,48]. The Pro-Pro-Ala motif may sterically hinder carboxypeptidase progression through the juxtaposed, bulky proline residues [47]. To assess the impact of addition of these motifs, mice were immunized with the ABKDgg or ABKDppa constructs (Fig.…”

Biotinylation Facilitates the Uptake of Large Peptides by Escherichia coli and Other Gram-Negative Bacteria

“…1 and 8 disappear overtime. This is due to degradation by peptidases and proteases that are present in bacterial cells (43). All studies were done in triplicate; however, only one representative Western blot is shown for each experiment.…”

UV-specific DNA repair recombinant fusion enzyme: A new stable pharmacologically active principle suitable for photoprotection

“…Using genetic engineering techniques we were able to produce aUveA, a fusion enzyme satisfying the need of a much more stable form of the UveA enzyme. This strategy for enzymatic stabilization has been already successful for other enzymatic activities [22,23]. Although fusion proteins were originally constructed to facilitate protein immobilization and purification, it has been demonstrated that certain peptides fused to proteins improve the stability and thereby the yield of recombinant protein expression [18].…”