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The dopamine 2 receptor (D2R) is a Class A GPCR which is essential for signaling in the nervous system, and has been implicated in numerous illnesses. While there are over 50 currently approved drugs which act on D2R, the structure has never been determined in detail. Although crystallography has historically been difficult with GPCRs, in recent years many structures have been solved using lipidic cubic phase (LCP) crystallization techniques. Sample preparation for LCP crystallization typically requires optimization of genetic constructs, recombinant expression, and purification techniques in order to produce a sample with sufficient stability and homogeneity. This study compares several genetic constructs utilizing different promoters, fusion proteins, fusion positions, and truncations in order to determine a high quality construct for LCP crystallization of
D2R. All constructs were expressed using the Bac-to-bac baculovirus expression system, then extracted with n-Dodecyl-β-D-Maltoside (DDM) and purified using metal affinity chromatography. Samples were then tested for quantity, purity, and homogeneity using SDS-PAGE, western blot, and size-exclusion chromatography. High quality samples were chosen based on insect cell expression levels, purification yield, and stability estimated by the levels of homomeric protein relative to aggregated protein. A final construct was chosen with which to continue future studies in optimization of thermal stability and crystallization conditions. Future work on this project is required to produce a sample amenable to crystallization. Screening of ligands for co-crystallization,
thermostabilizing point mutations, and potentially optimization of extraction and purification techniques prior to crystallization trials. Solving the D2R structure will lead to an increased understanding of its signaling mechanism and the mechanisms of currently approved drugs, while also providing a basis for more effective structure-based drug design.
D2R. All constructs were expressed using the Bac-to-bac baculovirus expression system, then extracted with n-Dodecyl-β-D-Maltoside (DDM) and purified using metal affinity chromatography. Samples were then tested for quantity, purity, and homogeneity using SDS-PAGE, western blot, and size-exclusion chromatography. High quality samples were chosen based on insect cell expression levels, purification yield, and stability estimated by the levels of homomeric protein relative to aggregated protein. A final construct was chosen with which to continue future studies in optimization of thermal stability and crystallization conditions. Future work on this project is required to produce a sample amenable to crystallization. Screening of ligands for co-crystallization,
thermostabilizing point mutations, and potentially optimization of extraction and purification techniques prior to crystallization trials. Solving the D2R structure will lead to an increased understanding of its signaling mechanism and the mechanisms of currently approved drugs, while also providing a basis for more effective structure-based drug design.
ContributorsErler, Maya Marie (Author) / Liu, Wei (Thesis director) / He, Ximin (Committee member) / School of Molecular Sciences (Contributor) / Barrett, The Honors College (Contributor)
Created2016-12
Description
The main objective of this project is to create a hydrogel based material system to capture and release CCRF-CEM Leukemia cancer cells via chemo-mechanical modulation. This system is composed of an aptamer-functionalized hydrogel thin film at the bottom of a microfluidic channel, which changes its film thickness as the temperature of the fluid in the system changes. The functionalized hydrogel film has been created as the primary steps to creating the microfluidic device that could capture and release leukemia cells by turning the temperature of the fluid and length of exposure. Circulating tumor cells have recently become a highly studied area since they have become associated with the likelihood of patient survival. Further, circulating tumor cells can be used to determine changes in the genome of the cancer leading to targeted treatment. First, the aptamers were attached onto the hydrogel through an EDC/NHS reaction. The aptamers were verified to be attached onto the hydrogel through FTIR spectroscopy. The cell capture experiments were completed by exposing the hydrogel to a solution of leukemia cells for 10 minutes at room temperature. The cell release experiments were completed by exposing the hydrogel to a 40°C solution. Several capture and release experiments were completed to measure how many cells could be captured, how quickly, and how many cells captured were released. The aptamers were chemically attached to the hydrogel. 300 cells per square millimeter could be captured at a time in a 10 minute time period and released in a 5 minute period. Of the cells captured, 96% of them were alive once caught. 99% of cells caught were released once exposed to elevated temperature. The project opens the possibility to quickly and efficiently capture and release tumor cells using only changes in temperature. Further, most of the cells that were captured were alive and nearly all of those were released leading to high survival and capture efficiency.
ContributorsPaxton, Rebecca Joanne (Author) / Stephanopoulos, Nicholas (Thesis director) / He, Ximin (Committee member) / Gould, Ian (Committee member) / Materials Science and Engineering Program (Contributor) / School of Molecular Sciences (Contributor) / Barrett, The Honors College (Contributor)
Created2016-12