Celiac disease is an autoimmune enteropathy resulting from the ingestion of gluten and gluten-like proteins from wheat, barley and rye in afflicted individuals. Indigestible gluten-derived peptides rich in proline residues are known to be responsible for eliciting the inappropriate immune response characteristic of the disease. In this investigation, surface level expression of prolyl peptidase activity by genetically engineered probiotic lactobacilli was postulated to be a possible treatment for this disease. Plasmid-based reporter vectors were constructed utilizing a novel, homology-based cloning technique to assess the expression and localization signals from the S-layer protein gene of Lactobacillus acidophilus. These plasmids were mutated during construction due to toxicity associated with the cloned cassettes. The toxicity of the Slayer secretion and/or anchoring domains in E. coli was confirmed by cloning the fused components into an inducible expression system. When the prolyl peptidase, Xaa-Pro, from L. reuteri was incorporated into the S-layer expression cassette, the full-length protein was efficiently expressed but was not active, likely due to protein aggregation and inclusion body formation. Future research directions are discussed and a modified experimental design strategy is presented. This work provides a foundation for continued investigation into the feasibility of utilizing genetically engineered lactobacilli as a potential treatment strategy for celiac disease.
| Identifer | oai:union.ndltd.org:CALPOLY/oai:digitalcommons.calpoly.edu:theses-1167 |
| Date | 01 June 2009 |
| Creators | Shurtleff, Matthew J |
| Publisher | DigitalCommons@CalPoly |
| Source Sets | California Polytechnic State University |
| Detected Language | English |
| Type | text |
| Format | application/pdf |
| Source | Master's Theses |
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