Inhibition of Eurygaster Integriceps Puton Prolyl Endoprotease (spPEP) and Human Prolyl Endopeptidase (hPEP) using αS1 ‐ Casein Peptide Inhibitors

Abstract

Prolyl endopeptidases (PEPs) are widely distributed among all domains of life (Di Cera., 2009). The focus of this study is mainly on prolyl endoprotease originated from the salivary glands of Erygaster integriceps Puton (Sunn pest) identified as spPEP by Darkoh (2008, 2010). The widely spread Eurygaster genus represents the most damaging and highly adaptable wheat pest in Northern Africa, Eastern Europe, Middle East with increasing concerns in Australia and United States. The natural substrate for spPEP is gluten from wheat grains, making spPEP the first known eukaryotic PEP capable of degrading substrates longer than 30 amino acid residues. To study the binding mechanism of this unique enzyme, recombinant peptide inhibitors were generated from αS1‐casein containing the inhibitory sequence LNENLLRFFVAPFPEV (Juillerat‐Jeanneret et al., 2011) varying in the length of the peptide and the positioning of the inhibitory sequence. All peptides were constructed with an N‐terminal 6x His Tag for purification as well as a Thrombin protease site for His Tag cleavage, ligated into pET15b vector and transformed into BL21(DE3)pLysS expression host (Vishram.,2016). The expression and purification of the peptides, including thrombin digestion and HPLC for final purification and quantification are shown. Enzyme kinetics for each of the recombinant inhibitory peptides in the presence of GPpNA as substrate is compared between spPEP and human prolyl endopeptidase (hPEP). This is the first study to show the hPEP to bind a peptide longer than 30 amino acid residues as well as inhibition of spPEP with large peptides.