Determination of matriptase-prostasin cleavage sites in the extracellular domain of the epidermal growth factor receptor (EGFR)
This year the American Cancer Society predicts that 565,650 individuals will lose their life as a result of their battle with cancer. Due to its established roles in cancer and extracellular presentation, the Epidermal Growth Factor Receptor (EGFR) is an excellent target for anti-cancer drugs. It has been determined that matriptase and prostasin serine proteases are proteolytic regulators of EGFR membrane presentation, and downstream signaling. Currently, there are several drugs that target EGFR, but research continues in order to further understand drug-resistant EGFR. In cancer cell lines that exhibit both EGFR signaling and these proteases, proteolytic cleavage may be a mechanism of resistance to drugs that target the EGFR extracellular domain (ECD). The specific aim of this project was to determine which protease was direct! y responsible for EGFR cleavage and establish the precise cleavage site within the EGFR ECD. DNA corresponding to amino acid residues 336-505 of the EGFR ECD was cloned into the p-GEX-6P-I vector and expressed as a GST-fusion protein in E.coli cells. This fusion protein was isolated and purified by affinity chromatography. Purified GSTEGFR BCD fusion protein was mixed with prostasin and matriptase and evaluated for cleavage. No cleavage was detected using this method. Trypsin serine protease was used to ensure the cleavability of the GST-EGFR ECD. The GST-EGFR ECD fusion protein was found to be inappropriate for determining matriptase or prostasin cleavage sites, which are now being pursued by other means.
This item is only available in print in the UCF Libraries. If this is your thesis or dissertation, you can help us make it available online for use by researchers around the world by downloading and filling out the Internet Distribution Consent Agreement. You may also contact the project coordinator Kerri Bottorff for more information.
Chai, Karl X.
Bachelor of Science (B.S.)
College of Medicine
Molecular Biology and Microbiology
Dissertations, Academic -- Medicine;Medicine -- Dissertations, Academic
Length of Campus-only Access
Honors in the Major Thesis
Weaver, Sarah Elizabeth, "Determination of matriptase-prostasin cleavage sites in the extracellular domain of the epidermal growth factor receptor (EGFR)" (2008). HIM 1990-2015. 761.