Development of a Prolyl Endopeptidase Expression System in Lactobacillus Reuteri to Reduce the Clinical Manifestation of Celiac Disease

Author

Kara Lynn Jew, California Polytechnic State University, San Luis ObispoFollow

DOI: https://doi.org/10.15368/theses.2019.80
Available at: https://digitalcommons.calpoly.edu/theses/2065

Date of Award

7-2019

Degree Name

MS in Biological Sciences

Department/Program

Biological Sciences

Advisor

Michael W. Black

Abstract

Celiac Disease (CD) is an autoimmune disorder that emerges due to the ingestion of gluten, a protein found in a variety of common grains such as wheat, rye, and barley. Approximately 1 in 100 individuals in the US suffer from CD, making it the most commonly diagnosed gastrointestinal disorder (Ciclitira et. al., 2005). These proline-rich gluten peptides are resistant to proteolysis and accumulate in the duodenum of the small intestine. Once in the duodenum, these peptides illicit an autoimmune response resulting in villous atrophy. Current treatment for CD requires a rigorous adherence to a gluten-free diet. Nevertheless, gluten-containing grains are ubiquitous in the western diet, so accidental

exposure to gluten remains as a persistent threat.

The approach of this project centers on genetically engineering a strain Lactobacillus reuteri to secrete a Myxococcus xanthus prolyl endopeptidase (PEP), an enzyme that hydrolyzes a peptide bond adjacent to an internal proline residue. The data from this study revealed that recombinant M. xanthus PEP purified from E. coli was effective in degrading Suc-Ala-Pro-pNA, a chromogenic substrate containing an internal proline residue. When introduced into a L. reuteri expression vector, mutations accumulated in the vector over the course of 5 days. These data suggested that toxicity was possibly associated with M. xanthus PEP and the amyl signal peptide.