Date of Award
Master of Science - Biotechnology
Dr. Beatrice Clack
Dr. Alexandra Van Kley
Dr. Rebecca Parr
Dr. Josephine Taylor
Eurygaster integriceps Puton, common name sunn Pest, is one of the primary sources of wheat crop wastes in North Africa, Middle East, and Eastern Europe. It feeds by injecting the wheat grain with an enzyme characterized as prolyl endoprotease (spPEP) that breaks down Gluten, the wheat’s main constitutive protein necessary for bread production (Darkoh et al., 2010). Previously, it has been shown that peptides isolated from Lactobacillus hydrolysates of caseins in bovine milk are able to inhibit mammalian PEP in colon cells, as well as bacterial PEP (Juillerat-Jeanneret et al., 2010). While recombinant versions of these peptides are also potential inhibitors of the spPEP, their specificity must be tested also against hPEP. The primary objective of this study was to clone hPEP into the same expression vector as spPEP in order to compare hPEP to spPEP with regards to substrate binding, recognition, and inhibition. Initially, hPEP was PCR amplified in order to incorporate the 5’ and 3’ ends necessary for ligation independent cloning (LIC) into the expression vector. This was then expressed in BL21(DE3)/pTFs and purified on a nickel column. Future studies will include comparing inhibition between hPEP and spPEP using 16 PCR amplified fragments of varying length that contain the non-allergenic (as demonstrated by Ruiter, 2005) inhibitory sequence LNENLLRFFVAPFPEVFG, isolated from bovine αS1 casein.
Moore, Travis K., "Cloning, Purification, and Biochemical Characterization of Human Prolyl Endopeptidase" (2017). Electronic Theses and Dissertations. 94.
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