Priprava in karakterizacija človeških rekombinantnih peptidnih fragmentov laktoferina in CD14 za vezavo na lipopolisaharide : doktorska disertacija
Majerle, Andreja
Recognition of components of bacterial envelope, notably lipopolysaccharides (LPS), is an important part of the system of innate immunity, which triggers response of immune cells. It leads to ...destruction of bacteria but it may also lead to septic shock, very often with fatal outcome. No effective drug againstsepsis exists at the moment. Knowledge about interactions on molecular level is essential for design of potential drugs, which could neutralize LPS. This led us to decision to investigate the interactions between proteins and peptides with LPS. CD14 has been identified as the high affinity cellular receptor for LPS. Binding of LPS to CD14 initiates the innate component of immune response and triggers a response that can lead to septic shock. In order to provide recombinant CD14 for the study of LPS-CD14 molecular interactions we have expressed functional fragments of human CD 14 in Escherichia coli and Pichia pastoris. In bacteria, we have prepared the full-length CD 14 and several shorter functional fragments of the protein, among them a CD14 fragment, consisting of the first Nterminal 152 residues, which retains the ability to bind LPS (Majerle et al., 2000č 1), (Majerle et al., 1999č 2). We have optimized the procedure for its refolding. An assay to monitor the refolding efficiency employing conformational specific anti-human CD14 monoclonal antibody has been established. Recombinant CD14 produced in yeast was glycosylated and secreted into the medium. We have shown that recombinant CD 14 from both bacteria and yeast bind to LPS with native electrophoresis gel shift and dot blot assays. Substances, which bind LPS, released from bacterial cell walls to the blood stream, can neutralize its toxic activity. Vertebrates, invertebrates, plants and fungi produce antimicrobial and endotoxin neutralizing proteins and peptides, which are usedas the first line of defence to light infection. (Abstract truncated at 2000 characters).
Type of material - dissertation
Publication and manufacture - Ljubljana : [A. Majerle], 2001
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