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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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