Time- and pH- dependent proteolysis of Engineered Proteins by Collagenase

Berry, Hunter (2019) Time- and pH- dependent proteolysis of Engineered Proteins by Collagenase. Undergraduate thesis, under the direction of Susan Pedigo from Chemistry & Biochemistry, The University of Mississippi.

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Abstract

Pleiotropic effects from oral administration of anti-inflammatory drugs limit their effectiveness. The ultimate goal of this project is to develop a novel self-assembling protein-based hydrogel for in situ delivery of NSAIDs at the site of chronic inflammation. Since inflammation causes acidification and activation of collagenases in the inflamed tissue, we have designed the hydrogel to respond to both of these queues to effectively deliver drug at the site. Self-assembly of the protein hydrogel exploits the high affinity and specific interaction between the protein calmodulin (CaM) and its specific binding peptide, M13. The two components of the hydrogel, CCLP and PCLP contain repeats of CaM and M13 domains, respectively, which are separated by engineered collagen-like-sequences (CLS). Proteolysis of the protein hydrogel formed by PCLP and CCLP by collagenases will cause exposure of the protomers and release of drug at the site. This important aspect of this responsive material requires susceptibility to relevant proteases, and is the specific subject of the present study. Exposure of CCLP to collagenase, and MMP-1, indicates that each protomer is susceptible to cleavage in the CLS sequences. This conclusion was confirmed by SDS-PAGE of cleaved CCLP. Further proof, through use of MALDI-TOF, was required in order to more precisely characterize fragment identity; however, lack of access to current working negative mode MALDI-TOF prohibited precise molecular weight quantification of fragment bands. Initial studies have prompted iterations of the component protomers. Further, we have demonstrated that the hydrogel is also susceptible to cleavage by collagenases. Results indicate the pH and Temperature dependent degradation of Engineered Proteins by collagenase require investigation into other degradable sequences as substitutes for current CLS to better improve lifetime in inflammatory conditions. Also, future research into kinetic profiles of the degradation of PCLP and CCLP by MMPs are necessary for optimizing the lifetime of the hydrogel for in-situ drug delivery.

Item Type: Thesis (Undergraduate)
Creators: Berry, Hunter
Student's Degree Program(s): B.A. in Biochemistry
Thesis Advisor: Susan Pedigo
Thesis Advisor's Department: Chemistry & Biochemistry
Institution: The University of Mississippi
Subjects: Q Science > QD Chemistry
Depositing User: Hunter Berry
Date Deposited: 16 May 2019 19:32
Last Modified: 17 May 2019 18:26
URI: http://thesis.honors.olemiss.edu/id/eprint/1540

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