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Structural Studies on Extracellular Domains of adhesion G Protein-Coupled Receptor ADGRG4
Strnadová, Martina ; Márová, Ivana (oponent) ; Mravec, Filip (vedoucí práce)
The aim of this thesis was to provide structural information on the huge extracellular region (ECR) of ADGRG4 that allows hypothesis of the function of the receptor. Therefore, the ECR was dissected into smaller, likely independently folded subunits. Subsequently, these samples were recombinantly expressed in human embryonic kidney 293 cells (HEK293S cells). These protein constructs were purified to allow for subsequent biophysical characterization. To increase the possibility of protein crystallisation, the purified protein was deglycosylated and then subjected to crystallisation trials. The second part of this thesis dealt with the analysis of the construct that contained the NTF part (the CTF was lacking), of well-established ADGRs. Ten different constructs were successfully cloned and expressed in HEK293T cells. Native PAGE and Dynamic Light Scattering (DLS) techniques were used to evaluate the results of the oligomeric state of the samples from both parts. As a control for autoproteolysis, which is a typical feature of aGPCR, various WB techniques were used. The Nano-Differential Scanner Fluorescence (DSF method was used to determine the prepared constructs and to find the optimal conditions for the buffers used in the thesis. The results of the first part show under which conditions the crystallisation trials were the most suitable that can be repeated and optimized further in the research. The second part showed which cloned constructs were able to be expressed in cells. Based on the parameters obtained from the first and second parts, research in this area can continue and, in the future, we can obtain important information about these remarkable receptors.
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