Date of Award

Spring 5-13-2017

Degree Type

Thesis

Degree Name

Master of Science - Biotechnology

Department

Biology

First Advisor

Dr. Beatrice Clack

Second Advisor

Dr. Alexandra Van Kley

Third Advisor

Dr. Rebecca Parr

Fourth Advisor

Dr. Josephine Taylor

Abstract

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.

Creative Commons License

Creative Commons License
This work is licensed under a Creative Commons Attribution-Noncommercial-No Derivative Works 4.0 License.

Available for download on Saturday, May 12, 2018

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