Honors Theses

Date of Award


Document Type

Undergraduate Thesis


Chemistry and Biochemistry

First Advisor

Susan Pedigo

Relational Format



Hydrogels have been explored for many biomedical applications, including targeted, in situ drug delivery to avoid the negative side effects associated with systemic delivery. In our work, we are exploiting the high affinity, calcium-dependent binding between calmodulin and its target peptides to create a biomaterial for in situ, extracellular drug delivery. Genes were engineered to make two protomers, Calmodulin Collagen-Like Protein (CCLP) and Peptide Collagen-Like Protein (PCLP), that will spontaneously self-assemble in situ due to the high Ca2+ concentration in the extracellular space and provide tunable, targeted drug delivery in a biocompatible hydrogel. One important factor that would dictate the potential utility and application of this hydrogel is the geometry of the protomers involved. Longer polymers have higher levels of entanglement and thus form gels with greater integrity. We used Thiol-Michael addition chemistry to bioconjugate our protomers with polyethylene glycol (PEG) crosslinkers in order to fine-tune the physical properties of the resultant hydrogel, such as elasticity and viscosity. We took advantage of the base-catalyzed Thiol-Michael addition mechanism and performed reactions between the cysteine residues of our genetically-engineered protomers and both divinyl sulfone and maleimide-based PEG crosslinking reagents. We studied the extent of bioconjugation and the effects of factors including concentration of reducing agent and denaturant, temperature, identity of the Michael acceptor, and the ratio of crosslinker to protein. Results were assayed by gel electrophoresis. We found that maleimide-PEG reagents are far more reactive than those based on divinyl sulfone, and at a lower pH. Additionally, bioconjugation is most promoted by the presence of 1 mM TCEP (reducing agent). Temperature and urea do not have significant effects on the reaction. The three constructs we tested were all modifiable, but we see particular potential in our CCLP Bis-Cysteine construct because it is capable of multiple bioconjugation reactions due to its bifunctional nature. The chemistry described here can be used to fine-tune the physical properties of the hydrogels formed by our protomers for a wide array of applications.

Accessibility Status

Searchable text

Included in

Biochemistry Commons



To view the content in your browser, please download Adobe Reader or, alternately,
you may Download the file to your hard drive.

NOTE: The latest versions of Adobe Reader do not support viewing PDF files within Firefox on Mac OS and if you are using a modern (Intel) Mac, there is no official plugin for viewing PDF files within the browser window.