Date of Award
Chemistry and Biochemistry
The goal of this work is to develop a self-assembling, protein-based gel to deliver drugs at the site of chronic inflammation. There are two engineered protomers that comprise the gel; calmodulin- and M13- are their critical components. Numerous enzymes have been shown to undergo calcium-dependent regulation by calmodulin, an intracellular protein highly resilient to its chemical environment. One of these enzymes, myosin light chain kinase, contains a remarkable 26 residue portion known as the M13 peptide that has been demonstrated to bind to calmodulin in the presence of calcium through hydrophobic interactions. Because the extracellular space in vivo constitutes an environment rich in calcium, mixtures of engineered proteins with M13 (called PMSP) and calmodulin (called CMSP) will form a network. These engineered protein mixtures can be functionalized with drugs for delivery to the extracellular space at sites of chronic inflammation. This two component binding system can be tuned to modify the affinity and cooperativity with which self-assembly occurs, thus making it the perfect vehicle for in situ drug delivery. The purpose of the work reported here is to study the M13 peptide-containing protomer, PMSP. Since this is a novel construct, this thesis reports basic studies of the bacterial expression, solubility, interactions with calmodulin, and evidence that it forms a gel with CMSP. In order to obtain PMSP sufficient for hydrogel formation, we have bacterially expressed the protein. We have found PMSP to express poorly, with yields increased upon expression from freshly transformed cells. Further, PMSP has limited solubility in aqueous solutions, requiring the addition of Guanidine HCl or DMSO to solubilize it. Once in solution, we found that the aqueous solubility of PMSP increased in the presence of free calmodulin implying that PMSP bound calmodulin. Secondly, PMSP appeared to bind to CMSP in the presence of calcium and to form a viscous solution. We are actively working to develop spectroscopic methods to support the apparent calmodulin-PMSP binding data. Finally, we will report our first attempts to measure the viscosity of the PMSP and CMSP mixture.
Carroll, Austin, "Assembly of a Protein-based Hydrogel as a Vehicle for In Situ Drug Delivery" (2018). Honors Theses. 104.