000125147 001__ 125147 000125147 005__ 20230322092647.0 000125147 037__ $$aTAZ-TFG-2022-3213 000125147 041__ $$aeng 000125147 1001_ $$aPedrós Manzanares, Laura 000125147 24200 $$aPurification of the Caseinolytic protease ClpP. 000125147 24500 $$aPurification of the Caseinolytic protease ClpP. 000125147 260__ $$aZaragoza$$bUniversidad de Zaragoza$$c2022 000125147 506__ $$aby-nc-sa$$bCreative Commons$$c3.0$$uhttp://creativecommons.org/licenses/by-nc-sa/3.0/ 000125147 520__ $$aThe caseinolytic protease P (ClpP) is an essential protein for bacteria and bacterial derived organelles. It belongs to the AAA+ family of proteases and has proteolytic activity, thereby, it plays an important role in protein digestion. This protease is involved in essential processes such as, cellular regulatory mechanisms, protein homeostasis, responses to environmental stimuli and host infections.<br />The ClpP complex is formed by two principal components. In one hand, two heptameric rings of ClpP forms the proteolytic core, with catalytic activity. The second major component is the Clp-ATPases, which forms an hexameric ring in both edges of the complex. The function of this ATPase is selecting the substrate for degradation. There are different Clp-ATPases depending on the organism, and each of them recognise a different substrate. For a further specificity, the Clp-ATPase interacts with adaptor proteins.<br />In humans, the only Clp-ATPase interacting with ClpP is ClpX. The human Clp system plays its role inside the mitochondrial matrix, therefore, a transport of both ClpP and ClpX to the mitochondrial matrix is required for the function of the protease. For this transport to happen, both proteins are first translated as premature proteins, with a Mitochondrial Target Sequence (MTS). This sequence will be then recognised by the cell machinery to transport the proteins inside the mitochondrial matrix. Afterwards, the MTS sequence will be cleaved when the proteins pass into the matrix.<br />In this study, the proteins of the human Clp system, as well as the Clp protease of S. aureus, were purified through the Äkta Column, using the Strep-tag purification technique. Prior to the purification, E. coli cells were transformed with a pET plasmid containing the gen of interest, and then the expression of the protein was induced with IPTG. Later, the purification through the column was performed.<br />Finally, after the purification, the protein concentration was measured, and the proteins were stored at -80°C for further in vitro assays. Afterwards, the purification results were also confirmed by doing an SDS-PAGE Electrophoresis.<br />As a conclusion, this study fathom in the understanding of Clp protease system expression in the cells, in order to use this knowledge in future studies with this complex.<br /><br /> 000125147 521__ $$aGraduado en Biotecnología 000125147 540__ $$aDerechos regulados por licencia Creative Commons 000125147 700__ $$aBrötz-Oesterhelt, Heike$$edir. 000125147 7102_ $$aUniversidad de Zaragoza$$b $$c 000125147 8560_ $$f774705@unizar.es 000125147 8564_ $$s1942088$$uhttps://zaguan.unizar.es/record/125147/files/TAZ-TFG-2022-3213.pdf$$yMemoria (eng) 000125147 909CO $$ooai:zaguan.unizar.es:125147$$pdriver$$ptrabajos-fin-grado 000125147 950__ $$a 000125147 951__ $$adeposita:2023-03-21 000125147 980__ $$aTAZ$$bTFG$$cCIEN 000125147 999__ $$a20220823110249.CREATION_DATE