Expression, Purification and Spectroscopic Analysis of L. helveticus PEPX and Triple Mutant
Faculty Sponsor
Deanna Ojennus, Whitworth University
Research Project Abstract
An x-prolyl dipeptidase (PEPX) is an enzyme that is capable of hydrolyzing gluten fragments at proline residues. Gluten causes a T-cell mediated response in the lining of the small intestine of Celiac Disease (CD) patients. Therefore, CD patients must adhere to a gluten free diet. The presence of PEPX supplemental enzymes in the digestive tract of a CD patient may allow for digestion of gluten. However, PEPX enzymes must be pepsin resistant in order to maintain activity in the small intestine. A modified PEPX (PEPX 3M) contains three mutations designed to remove high frequency pepsin cut sites (F133W, W161Q, L684H) and is a potential candidate for an oral therapeutic. However, these mutations were found to decrease pepsin resistance when compared to w.t. PEPX. Stability of the 3M mutant was considered as a possible explanation for its lack of pepsin resistance. The PEPX 3M was expressed in E. coli and purified via affinity and ion exchange chromatography. Temperature denaturation analysis via circular dichroism spectropolarimetry showed a lack of thermal stability in the PEPX 3M enzyme when compared to the w.t. PEPX.
Session Number
PS1
Location
Graves Gym
Abstract Number
PS1-o
Expression, Purification and Spectroscopic Analysis of L. helveticus PEPX and Triple Mutant
Graves Gym
An x-prolyl dipeptidase (PEPX) is an enzyme that is capable of hydrolyzing gluten fragments at proline residues. Gluten causes a T-cell mediated response in the lining of the small intestine of Celiac Disease (CD) patients. Therefore, CD patients must adhere to a gluten free diet. The presence of PEPX supplemental enzymes in the digestive tract of a CD patient may allow for digestion of gluten. However, PEPX enzymes must be pepsin resistant in order to maintain activity in the small intestine. A modified PEPX (PEPX 3M) contains three mutations designed to remove high frequency pepsin cut sites (F133W, W161Q, L684H) and is a potential candidate for an oral therapeutic. However, these mutations were found to decrease pepsin resistance when compared to w.t. PEPX. Stability of the 3M mutant was considered as a possible explanation for its lack of pepsin resistance. The PEPX 3M was expressed in E. coli and purified via affinity and ion exchange chromatography. Temperature denaturation analysis via circular dichroism spectropolarimetry showed a lack of thermal stability in the PEPX 3M enzyme when compared to the w.t. PEPX.
