Exploration of Thiol-Michael Addition Bioconjugation to Extend Polymers of a Protein-Based Hydrogel

Smith, Elaine Wallin (2019) Exploration of Thiol-Michael Addition Bioconjugation to Extend Polymers of a Protein-Based Hydrogel. Undergraduate thesis, under the direction of Susan Pedigo from Chemistry & Biochemistry, University of Mississippi.

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Abstract

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.

Item Type: Thesis (Undergraduate)
Creators: Smith, Elaine Wallin
Student's Degree Program(s): B.A. Biochemistry
Thesis Advisor: Susan Pedigo
Thesis Advisor's Department: Chemistry & Biochemistry
Institution: University of Mississippi
Subjects: Q Science > QD Chemistry
Depositing User: Elaine Smith
Date Deposited: 10 May 2019 04:48
Last Modified: 10 May 2019 04:48
URI: http://thesis.honors.olemiss.edu/id/eprint/1415

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