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Preparation of neprosin I - unique aspartic protease with potential in structural biology, proteomics and treatment of gluten intolerance.
Pelechová, Kamila ; Man, Petr (advisor) ; Šmídová, Aneta (referee)
Proteases are being found in all realms from viruses and microorganisms to plants and animals, including humans. They are necessary for living organisms. They degrade proteins to peptides and amino acids, allowing organisms to better absorb nutrients. Proteases also have a regulatory function - they control the cell cycle, they are responsible for converting precursors to biologically active substances, they are part of signaling cascades. Thanks to their properties, proteases are used for therapeutic purposes but found their used also in industry and research. This work deals with the production of recombinantly prepared neprosin - a newly discovered prolyl endopeptidase isolated from the carnivorous plant Nepenthes ventrata. It is an acidic protease with potential for use in structural biology, proteomics and medicine. Due to its ability to cleave primarily C-terminal proline residues, neprosin become a promising option for celiac enzyme therapy. (In Czech)
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Preparation of proteases for protein structure studies.
Jirečková, Barbora ; Man, Petr (advisor) ; Vaňková, Pavla (referee)
Hydrogen/deuterium exchange coupled with mass spectrometry (HDX-MS) is a method allowing the study of protein structure and dynamics. Its spatial resolution is given by the proteolysis step that is included in the HDX-MS workflow. Most widely used pepsin has however some limitations and use of a single protease often does not provide optimal spatial resolution. Several publications have emphasized the importance of the alternative proteases nepenthesin-2 (Nep-2) and aspergillopepsin (XIII) cleaving, in contrast to pepsin, after basic amino acids. In studies targeting proline-rich proteins, another enzyme, prolyl endoprotease from Aspergillus niger (AnPEP), is gaining importance. This work focuses on the characterization of immobilized AnPEP in combination with pepsin, aspergillopepsin or Nep-2 for their application in HDX-MS. First, columns with only one protease were tested on a set of model proteins. It was found that immobilized AnPEP did not have optimal cleavage characteristics compared to the other proteases. In order to combine the advantages of the proteases mentioned above, the model proteins were digested using columns with AnPEP coimmobilized with pepsin, Nep-2 or XIII and also using two protease columns in series (always AnPEP column with pepsin, Nep-2 or XIII column in both...
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Eukaryotic expression of acid proteases.
Vodolánová, Lucie ; Man, Petr (advisor) ; Pavlíček, Jiří (referee)
Nepenthesin I and neprosin are acid proteases which are present in the pitcher fluid of carnivorous plants genus Nepenthes. Both enzymes are produced as zymogens which are activated in acidic conditions. Due to their properties and specific preferences, they are suitable for protein mass spectrometry or as a supplement in gluten-free diet of celiac patients. Quantity of the enzymes in the pitcher fluid is very low and the currently developer expression protocols do not show satisfactory results, we tried to find more suitable expression system. We would like to produce high quantitites of enzymes, which will have the same stability as natural ones. The aim of this work is preparation of expression vectors with nepenthesin I and neprosin genes for transfection into eukaryotic expression systems of S2 and HEK 293 cells. In Czech. Keywords: acid proteases, nepenthesin I, neprosin, expression vector
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Eukaryotic expression of acid proteases.
Vodolánová, Lucie ; Man, Petr (advisor) ; Pavlíček, Jiří (referee)
Nepenthesin I and neprosin are acid proteases which are present in the pitcher fluid of carnivorous plants genus Nepenthes. Both enzymes are produced as zymogens which are activated in acidic conditions. Due to their properties and specific preferences, they are suitable for protein mass spectrometry or as a supplement in gluten-free diet of celiac patients. Quantity of the enzymes in the pitcher fluid is very low and the currently developer expression protocols do not show satisfactory results, we tried to find more suitable expression system. We would like to produce high quantitites of enzymes, which will have the same stability as natural ones. The aim of this work is preparation of expression vectors with nepenthesin I and neprosin genes for transfection into eukaryotic expression systems of S2 and HEK 293 cells. In Czech. Keywords: acid proteases, nepenthesin I, neprosin, expression vector
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Preparation of neprosin I - unique aspartic protease with potential in structural biology, proteomics and treatment of gluten intolerance.
Pelechová, Kamila ; Man, Petr (advisor) ; Šmídová, Aneta (referee)
Proteases are being found in all realms from viruses and microorganisms to plants and animals, including humans. They are necessary for living organisms. They degrade proteins to peptides and amino acids, allowing organisms to better absorb nutrients. Proteases also have a regulatory function - they control the cell cycle, they are responsible for converting precursors to biologically active substances, they are part of signaling cascades. Thanks to their properties, proteases are used for therapeutic purposes but found their used also in industry and research. This work deals with the production of recombinantly prepared neprosin - a newly discovered prolyl endopeptidase isolated from the carnivorous plant Nepenthes ventrata. It is an acidic protease with potential for use in structural biology, proteomics and medicine. Due to its ability to cleave primarily C-terminal proline residues, neprosin become a promising option for celiac enzyme therapy. (In Czech)
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