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Reaction of prion protein with quinacrine
Zawada, Zbigniew ; Šafařík, Martin ; Šebestík, Jaroslav ; Stibor, Ivan ; Bouř, Petr
Prion protein is supposed to cause Creutzfeld–Jakob disease, which can be treated in cell culture by quinacrine. In this work we study acridinylation reaction of prion protein. We also investigate this reaction theoretically on simple models and calculate activation energies for a large ensemble of acridine derivatives. Found correlation between the reactivity and geometry can enhance the design of the new acridine compounds, particularly for medical purposes.
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Expression of osmotin, an antifungal protein from Nicotiana tabacum in Escherichia coli
Viktorová, J. ; Macková, M. ; Macek, Tomáš
Plants have evolved a huge variety of proteins involved in the defense against pathogens and adaptation to stressful environments. Plant proteins whose expression is strongly induced in response to infection by pathogens belong to the group of pathogenesis-related (PR) proteins. The family of PR-5 proteins constitutes a group of cysteine-rich proteins including thaumatin, zeamatin and also osmotin. Osmotin is a cationic protein of 205 residues and molecular weight of 24 kDa. It was discovered and characterized in cells of Nicotiana tabacum var. Wisconsin 38. The plasmid harbouring cDNA of osmotin from Nicotiana tabacum was constructed for transformation of Escherichia coli. The osmotin gene was prepared in fusion with histidine tail to facilitate the isolation and purification from bacterial cells. Selection of transgenic colonies was based on antibiotic resistance. The hexahistidine-tagged osmotin was overexpressed in heterologous system by using pET expression vector and purified using immobilized metal affinity chromatography. The expression of osmotin was detected and antifungal activity was tested.
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Synthetic scan of C-domain from prion proteins
Šebestík, Jaroslav ; Zawada, Zbigniew ; Šafařík, Martin ; Hlaváček, Jan
Prions are suspected as culprits of several neuropathogenic diseases. A combination of chemical and recombinant syntheses is a powerful tool for studying prion role in pathogenesis of neurodegenerative diseases. Due to the presence of “difficult” sequences in the prion protein molecule, chemical ligation procedure using peptide thioesters as key building blocks is a method of choice. Therefore, a synthetic scan of peptides constituting the mouse prion protein (MoPrP) C-domain 93-231 was carried out. The synthesis on chlorotritylchloride resin was the most promising for most peptides. Only the octadecapeptide MoPrP195-212 could not be synthesized by automated peptide synthesis even using the β-sheet breaking dipeptides. This difficulty was overcome by manual peptide synthesis with careful monitoring of coupling and Fmoc removal.
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Study of antimicrobial peptide induction in Brassica napus
Shevchenko, I. ; Neubauerová, Tereza ; Macková, Martina ; Macek, Tomáš
Development of cheap, effective and environmentally friendly protection of industrial plants is a key problem of mankind, where using of antimicrobial peptides could be a reasonable solution. As a source for isolation of these peptides canola (Brassica napus) was chosen, which was treated by functional analog of salicylic acid – benzothiadiazole (BTH) – to stimulate the immune system. To isolate the responsible antimicrobial peptides, crude buffer extract was obtained. Next steps were precipitation, centrifugation and molecular weight filtration. The further isolation procedure comprised hexane extraction, SPE-C18 (Solid Phase Extraction) to separate hydrophobic and hydrophilic fractions and reversed-phase chromatography. Antimicrobial activity was tested by diffusion method. Fractions were tested on gram-positive and gram-negative bacteria and on yeast Candida scotii.
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Isolation and study of the properties of recombinant pseudoosmotin
Richterová, K. ; Nováková, Martina ; Antošová, Z. ; Neubauerová, Tereza ; Macková, Martina
Antimicrobial peptides represent new clinical compounds with antimicrobial action and they could participate in solving the problem of ever-increasing resistance of microorganisms to the traditional antibiotics. The aim of this work was to study optimal conditions for expression, isolation and purification of recombinant pseudoosmotin from Escherichia coli BL21 (DE3). Pseudoosmotin was isolated from membrane fraction of bacteria and was purified by the affinity chromatography using Ni-NTA. Isolated pseudoosmotin did not show antimicrobial activity against studied microorganisms.
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Larvae of flesh fly Neobellieria bullata as a source for novel antimicrobial peptides
Neubauerová, Tereza ; Macková, Martina ; Macek, Tomáš ; Šanda, Miloslav ; Voburka, Zdeněk
The antibiotic resistant strains of microorganisms are increasing threat for public health. Antimicrobial peptides from natural sources display a wide spectrum of activity. In our laboratory, several peptide fractions with antimicrobial acivity against gram-positive and gramnegative bacteria and fungi were isolated from larvae of flesh fly Neobellieria bullata. The presence of peptides was proved by tricine electrophoresis, molecular weights and sequences determination by MS and Edman sequencing, respectively. In comparison with database sequences we obtained structures present mainly in insect and amphibian antimicrobial peptides, but total matches were not higher than in 36%. Upon the obtained sequence motifs we designed structures of several synthetic peptides. These were synthetised on the Rink amide MBHA resin. Synthesis was performed according Fmoc/PyBOP-DIEA protocol. Antimicrobial activity of these peptides was determined.
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Isolation of antimicrobial peptides and proteins from tomato
Králová, M. ; Šanda, Miloslav ; Macková, M. ; Macek, Tomáš
Recent emerging problem is occurrence of multiresistant pathogens. This inconvenience makes people to search for new antimicrobials. One of possible ways is looking for natural antimicrobial peptides. In our project, green tissues of tomato (Solanum lycopersicum) were chosen for isolation of such peptides. Extracts obtained were fractionated by solid phase extraction and liquid chromatography. Antimicrobial activities were tested on model microorganisms. Several fractions exhibited antimicrobial activity against bacteria and fungi. In the extracts, precursors of two antifungal proteins were identified.
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