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Posttranslational modifications in soluble recombinant therapeutical proteins secreted by lower aukaryotes: structure and function
Kumar, Vinay ; Bezouška, Karel (advisor) ; Šedo, Aleksi (referee) ; Grubhoffer, Libor (referee)
Virtat, Kunnr M.Sc Aint of the stud1, Aim of the studv The aim of this study was to understand the molecular mechanisnrs contributing to the production of soluble leukocyte receptors in the eukaryotic expression systenrs including the proper posttranslational modifications such as disulfide bond fornration and glycosylation. In order to achieve this goal, the following specific aims have been adopted for the study: l, t. To develop new methodologies for rapid and convenient assessnrent ofthe disulfide L I boltds in complex eukaryotic proteins using SDS polyacrylarride electrophoresis under nonreducing conditions, arrd nrass spectrometry. 2. To establish elements that are critical for the stability of soluble CD69 receptors expressed in the bacterial expression systenr 3. To develop methods for the production of glycosylated soluble CD69 proteins in lower eukaryotes (P i c h i a pa s to ri s) 4. To purify the proteins in sufficient quantities for biochemical and inrnrunological studies 5. To investigate, how disulfide bonding and glycosylation influeuces the stability of soluble CD69 receptors 6. To look at the carbohydrate binding activities ofthe produced proteins. 7. To evaluate the in vivo propefties of the produced proteins including their circulation half lifes in the blood of experiental animals, and...
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The Study of the Immune Response of Larvae of the Fleshfly Sarcophaga Bullata
Mášová, Alice ; Jiráček, Jiří (advisor) ; Žurovec, Michal (referee) ; Bezouška, Karel (referee)
Conclusion lnsect immune response is a complicated process. Antimicrobiď peptides fiequently act synergistically, some proteins have transporter functions, peptide inhibitors can block processing enzyrnes involved in activating cascades or different enzymes can activarc antimicrobially active proteins or peptides. In this study we presented nvo different isolation protocols, which resulted in the identification of several already known and two novel antirnierobial proteins or peptides from the hemolymph of the larvae of flesbfty. sareophaga bullan. We are the first group to monitor the time<ourse of tissue-specific expÍession patlems of eight genes in Sarcophaga bullan |arvae ďter different immune challenges using qPCR. we show similarities, as well as differences in insect immune responses. we are also the first group to analyze the expression pattoms of the genes that encode sBp and sarcocystatin, proteins that are mainly connect€d with larvat development and metamorphosis. Using 2D- electrophoresis we analyzď the time.dependent immune response in larval fat bodies and hemocytes. We detect€d 9 up.regulated proteins in hemocyŮes and 15 differentially expressed proteins in fat body cells. We hope that our study will shed more light into ttre complex processes of immune responses in Sarcophaga bullan larvae. 20
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Interaction of the adenylate cyclase toxin with complement receptor 3 - Relation of structure and function
Morová, Jana ; Šebo, Peter (advisor) ; Konvalinka, Jan (referee) ; Bezouška, Karel (referee)
4 CoNcl-usroxs PnonucrroN oF tNrEcRtN CDI lb/CDlg F Four fragments of the subunit cDilb were produced and purified and then they were used for immunization of mice and for preparation of specific antibodíes. detected that 52 cells are not able to transport effectively this subunit to cell surface or to secrete its extracellular domain into medium and not even it has a signal peptide specific for 52 cells. Further it has been shorvn, that the level of production of CDl8 subunit was not affected by usage of constitutive or inducible plasmid. ANALYSIS oF THE |NTERÁCTIoN oF RTx ToxINs w|TH p2 INTEGRINS - THE RoLE oF THT, GLYCOSYLATION OF RECEPTORS IN BINDINC ON RTX TOXIN of cell surface glycoproteins, suggesting that cyaA binding to the cell surface- expressed cDllb/cDl8 integrin fully depends on its glycolsylation. It has been a|so demonstrated. that the deglycosylation did not affected ťormation of CD I I b/CD l8 heterodimer or its expression to cell surface. inhibited in the presence ofonly saccharide units that occur in the oligosaccharide chain of integrin molecule. This demonstrates, that cyaA directry recognizes the N-linked oligosaccharide chain ofits p2 integrin recepror. CD l I b/CD I 8-expressing cells. cytotoxic activity of others RTX toxins. l5...
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Analysis of influence of glycosylation on assembly and molecular architecture of enzymes.
Horáková, Jana ; Bezouška, Karel (advisor) ; Ryšlavá, Helena (referee)
Our laboratory studies the influence of glycosylation on protein architecture and the biological activity of fungal hexosaminidases. I studied the enzyme from Penicillium oxalicum which has an advantage of quite a homogenous glycosylation. The propeptide of this enzyme is naturally glycosylated only at two places - asparagine 11 and serine 66. Catalytic subunit and propeptide of the hexosaminidase enzyme from Aspergillus oryzae were separated from each other for reconstitution experiments. In the propeptide part of the enzyme separated from the Penicillium oxalicum hexosaminidase, asparagine 11 and serine 66 were swapped for cysteine by targeted mutagenesis. The newly synthesized propeptide modified with cellobiose, which is a compound most similar to naturally occurrency saccharide chitobiose, promised a significant chance of restoration of enzyme activity. The glycosylated propeptide was separated from the nonglycosylated. In reconstitution experiments we observed the influence of glycosylation on the enzyme structure and activity using different glycosylated propeptides. The highest efficiency occurred during the reconstitution by the original propeptide. Next we used combinations of artificially glycosylated propeptides of which the highest reconstitution efficiency had cellobiose(Asn11...
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α-N-Acetylgalactosaminidase as a tools in the synthesis of complex oligosaccharide immune stimulators
Mrázek, Hynek ; Bezouška, Karel (advisor) ; Macek, Tomáš (referee) ; Wimmerová, Michaela (referee)
1 Charles University in Prague Faculty of Science Department of Biochemistry α-N-Acetylgalactosaminidase as a tools in the synthesis of complex oligosaccharide immune stimulators Summary of Ph. D. Thesis Mgr. Hynek Mrázek Supervisor: Prof. RNDr. Karel Bezouška, Dsc. Prague 2011 2 Introduction Glycoproteins Glycoproteins consist of proteins to which carbohydrate are covalently linked. The distinction between proteoglycans and glycoproteins residues is in the level and types of carbohydrate modification. Carbohydrates are linked to the protein component through either O-glycosidic or N-glycosidic bonds. The N-glycosidic linkage is through the amide group of asparagine. The O- glycosidic linkage is to the hydroxyl of serine, threonine or hydroxylysine. The predominant carbohydrate attachment in glycoproteins of mammalian cells is via N-glycosidic linkage. The site of carbohydrate attachment to N- linked glycoproteins is found within a consensus sequence of amino acids, N-X- S(T), where X is any amino acid except proline. While the N-glycosylation is governed by the above rules, no exact rules were found for glycosylation of O- type. This process is the step-wise addition of the sugar residues directly onto the polypeptide chain. Biosynthesis of glycoproteins occurs via protein glycosylation (the addition of...
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Recombinant expression and studies on DCL-1, receptor of dendritic cells
Pospíšilová, Eliška ; Bezouška, Karel (advisor) ; Novák, Petr (referee)
Recombinant expression and studies on DCL-1, receptor of dendritic cells Eliška Pospíšilová Charles University in Prague, Faculty of Science, Department of Biochemistry ABSTRACT Dendritic cells, which could be found in large numbers in many tissues, exprime C-type lectine receptor DCL-1 (in CD nomenclature CD302) on their surface. This molecule belongs to the class one transmembrane proteins. N-terminal sequence a C-type lectin-like domain (CTLD) it has on the extracellular side. Whereas the C-terminus, which contains potential phosphorylation site and signals for intracellular transport, is in the cytosol. As the sequence of this protein is highly conserved through all mammalian species, it is likely to play an important role in the immune system. Despite this fact, the DCL-1 molecule is still poorly investigated. This work deals with the extracellular part - especcially with the CTLD containing part - of the DCL-1 receptor. To learn more about this molecule, we attempted its recombinant expression in bacteria. We used pET-30a(+) based vector pDCL1E, and bacterial cells E. coli BL21 (DE3) Gold, which produced the target protein in the form of inclusion bodies into the cytoplasm. Therefore conditions for in vitro renaturation were optimalized, and it was prooved that the protein had native conformation...
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