| TAZ-TFG-2022-3213 |
Purification of the Caseinolytic protease ClpP.
Pedrós Manzanares, Laura
Brötz-Oesterhelt, Heike (dir.)
Universidad de Zaragoza,
CIEN,
2022
Graduado en Biotecnología
Resumen: The 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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
Tipo de Trabajo Académico: Trabajo Fin de Grado
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