Purification Protocol Development and Analysis of Binding Properties of Calmodulin Collagen-Like Protein

Boddie, Patrick P (2018) Purification Protocol Development and Analysis of Binding Properties of Calmodulin Collagen-Like Protein. Undergraduate thesis, under the direction of Susan Pedigo from Chemistry & Biochemistry, The University of Mississippi.

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Calmodulin is an intensely studied, intracellular, calcium-binding protein that is involved in diverse physiological processes. When bound to calcium, calmodulin undergoes a conformational change that allows it to bind specific target peptides with high affinity, one being the calmodulin binding region of myosin light chain kinase, herein called M13. This high affinity, calcium dependent association has made it a desirable choice for use in a biomaterial for drug delivery for non-steroidal anti- inflammatory drugs (NSAIDs). Two artificial genes have been designed by our lab for use in the proposed biomaterial: calmodulin collagen-like protein (CCLP), a construct composed of two calmodulin genes connected with a gene coding for three repeats of a short collagen sequence; and PCLP, a construct composed of two repeats of M13 connected with same collagen repeats. Mixture of the two recombinant protein products should form a gel in the high extracellular calcium concentration found in vivo. In order to establish the viability of this novel biomaterial, we had to establish 1) protocols for expression and purification of CCLP, and 2) whether or not the calmodulin regions of CCLP retained their calcium-dependent binding of M13. In order to proceed with experimentation, a consistent protocol for the production and purification of the CCLP protein was developed. Confirmation of purity and concentration was obtained with SDS- PAGE and UV-Visible spectroscopy, respectively. Second, because M13 undergoes a change in secondary structure upon binding, CCLP’s interaction with M13 was monitored via circular dichroism spectroscopic studies. Results of these studies indicate that we have an efficient expression and purification protocol for CCLP, and that CCLP binds calcium and M13 according to its design.

Item Type: Thesis (Undergraduate)
Creators: Boddie, Patrick P
Student's Degree Program(s): BA Biochemistry
Thesis Advisor: Susan Pedigo
Thesis Advisor's Department: Chemistry & Biochemistry
Institution: The University of Mississippi
Subjects: Q Science > QD Chemistry
Depositing User: Mr. Patrick P Boddie
Date Deposited: 17 May 2018 21:50
Last Modified: 17 May 2018 21:50
URI: http://thesis.honors.olemiss.edu/id/eprint/1236

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