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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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Characterization and application of proline-selective proteases.
Portašiková, Jasmína Mária ; Man, Petr (advisor) ; Kádek, Alan (referee)
Peptide bond formed by proline is resistant to most known proteases, therefore the discovery and isolation of proteases cleaving after this amino acid opens up new possibilities not only for protein characterization but also for industrial, food or pharmaceutical aplications. Aspergillus niger prolyl endopeptidase (AnPEP) is a proline-selective protease that is commercially available in a variety of products. In this bachelor thesis cleavage preferences of proteases AnPEP (Clarity Ferm) and ProAlanase (Promega) were characterized and compared. The effect of different conditions on the specificity of proteases, while cleavaging the mixture of model proteins was examined within this work. AnPEP cleavage preferences were characterized on complex protein mixtures and its immobilized form was tested as well. [IN CZECH] Keywords: ProAlanase, AnPEP, H/D exchange, structural proteomics, imobilized proteases [IN CZECH]
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Proteomická a bioinformatická charakterizace N-terminálních sekvencí proteinů modifikovaných po importu do hydrogenosomu Trichomonas vaginalis.
Zákoucká, Eva ; Man, Petr (advisor) ; Šulc, Miroslav (referee)
Trichomonas vaginalis is a human pathogen causing trichomoniasis, one of the most common non-viral sexually transmitted diseases in both men and women. Trichomoniasis is currently treated with metronidazole, but the pathogen is known to develop resistance against this drug. However as the pathogen is eukaryotic, the targets for the pathogen elimination without seriously affecting the host are limited. Throughout the evolution Trichomonas vaginalis adapted to anaerobic environments by developing an alternative metabolism resulting in a reduced form of mitochondria named hydrogenosome. Hydrogenosomes lack genetic information, therefore all its proteins are nucleus-encoded and need to be transported inside the hydrogenosome using a targeting N-terminal presequence. The peptidase recognizing and cleaving those presequences at the entrance of the organelle, the hydrogenosomal processing peptidase (HPP), is unique for hydrogenosomes and therefore represents a potential drug target against the pathogen. In this work the HPP's substrate specificity towards the targeting presequences was investigated. To do so a proteomic analysis of the proteome of Trichomonas vaginalis hydrogenosomes was performed using a novel optimized protocol for N-terminal peptide sequencing. N-terminal peptides were captured using a...
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Application of acid proteases from nepenthes in hydrogen/deuterium exchange.
Darebná, Petra ; Man, Petr (advisor) ; Stráňava, Martin (referee)
Application of acid proteases from Nepenthes in hydrogen/deuterium exchange Petra Darebná (Department of Biochemistry, Faculty of Science, Charles University in Prague, Czech Republic) Nepenthes are mostly found in Borneo and Sumatra. They are one of a few carnivorous plants which produce its own proteolytic enzymes (nepenthesin I and nepenthesin II), which provide an alternative source of nitrogen and other nutrients in case that these plants grow in a soil which lacks such nutrients. These aspartate proteases are capable of proteolysis at a very low pH of a digestive gastrovascular cavity fluid after catching insects and in a cooperation with other proteins participating in digestion process. Processing of a digestive fluid, isolated from a digestive gastrovascular cavities of carnivorous plants of the Nepenthes genus and its possible application as a tool in a protein study using hydrogen/deuterium exchange were done in this thesis. Isolates of the digestive fluids were purified from coarse-grained impurities by centrifugation, activated by acidification and concentrated by ultrafiltration. The amount of proteins and their protein profile were monitored and an activity of acidic proteases was determined by enzymatic assay. Consequently, using LC-MS/MS and model proteins, the cleavage preferences...
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Preparation and use of acid proteases for digestion in H/D exchange.
Kukla, Jan ; Man, Petr (advisor) ; Pompach, Petr (referee)
- 5 - Abstract Hydrogen/deuterium exchange coupled to mass spectrometry (HX-MS) utilizes the spontaneous exchange of protein backbone amide hydrogens for deuterium atoms from solution to gain information about changes in protein structure. To localize these changes to specific areas of the protein, enzymatic digestion by aspartate proteases is used. The proteases' ability to produce small overlapping peptides and to provide full sequence coverage of the studied protein is essential for pinpointing the protein regions of interest. In this study recombinant proteases nepenthesin I (Nepenthes gracilis) and rhizopuspepsin (Rhizopus chinensis) were prepared and compared to commercially available proteases porcine pepsin A and aspergillopepsin (Aspergillus saitoi). The comparison was performed using various activity assays, where the effects of pH, temperature and denaturing and reducing agents on the activity of the proteases were studied. All four proteases were also immobilized on a polymeric resin POROS and their activity in an online HX-MS digestion setup was tested using myoglobin as a model substrate.
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Expression of recombinant soluble form of NKR-P1A receptor.
Vaňková, Pavla ; Man, Petr (advisor) ; Mrázek, Hynek (referee)
NK cells are large granular lymphocytes and they are part of the innate immune system. In defense against tumor or virus-infected cells they activate their cytotoxic mechanisms and by production of cytokines NK cells can mobilize assistance in form of adaptive immune response. They create a large area of interest because of finding of ligands to their receptors and understanding these interactions can lead to significant discoveries. In particular, in medicine, where it can be applied i.a. for transplantation, in the fight against tumors or HIV. This thesis focuses on NKR-P1A receptor, which belongs to NKR-P1 family with other six receptors. This protein is activation receptor and recently its novel isoform was discovered. New isoform differs in transmembrane domain, whose considerable part is lacked. Aim of the thesis was prepare production plasmid encoding mNKR-P1A ISO2 followed by test of expression.
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Study of receptor-ligand pair NKR-P1F and Clrg
Kotýnková, Kristýna ; Man, Petr (advisor) ; Schneider, Bohdan (referee)
Study of receptor-ligand pair NKR-P1F and Clrg Mouse NKR-P1F:Clr-g receptor:ligand pair is important component of the receptor "zipper" that occurs at the contact between natural killer cell and its target cell, and represents a recently discovered example of lectin-lectin interactions important for recognition among immune cell subsets. In order to study structure of these proteins and interactions between them, we have prepared pET-30a(+) bacterial expression vectors coding parts of extracellular domains of the two receptors. After induction of protein production with IPTG, the proteins precipitated into inclusion bodies, from which they could be refolded in vitro. Refolded proteins were purified using combination of ion exchange and size exclusion chromatography. NKR-P1F construct yielded only small amounts of soluble protein using standard refolding protocols. Furthermore we have experienced difficulties with reproducibility of the refolding results. In the case of Clrg the standard protocols for protein refolding were not sufficient. In order for the Clrg to fold properly, the odd cysteine which does not fit into the pattern usual for this family of receptors was substituted for serine and resulting C148S construct was shown to be more useful. Further, using (benzyldimethylammonio)propanesulfonate in...
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