Dokument-ID Dokumenttyp Verfasser/Autoren Herausgeber Haupttitel Abstract Auflage Verlagsort Verlag Erscheinungsjahr Seitenzahl Schriftenreihe Titel Schriftenreihe Bandzahl ISBN Quelle der Hochschulschrift Konferenzname Quelle:Titel Quelle:Jahrgang Quelle:Heftnummer Quelle:Erste Seite Quelle:Letzte Seite URN DOI Abteilungen OPUS4-2722 Dissertation Oey, Melanie Chloroplasts as bioreactors : high-yield production of active bacteriolytic protein antibiotics Plants, more precisely their chloroplasts with their bacterial-like expression machinery inherited from their cyanobacterial ancestors, can potentially offer a cheap expression system for proteinaceous pharmaceuticals. This system would be easily scalable and provides appropriate safety due to chloroplasts maternal inheritance. In this work, it was shown that three phage lytic enzymes (Pal, Cpl-1 and PlyGBS) could be successfully expressed at very high levels and with high stability in tobacco chloroplasts. PlyGBS expression reached an amount of foreign protein accumulation (> 70% TSP) that has never been obtained before. Although the high expression levels of PlyGBS caused a pale green phenotype with retarded growth, presumably due to exhaustion of plastid protein synthesis capacity, development and seed production were not impaired under greenhouse conditions. Since Pal and Cpl-1 showed toxic effects when expressed in E. coli, a special plastid transformation vector (pTox) was constructed to allow DNA amplification in bacteria. The construction of the pTox transformation vector allowing a recombinase-mediated deletion of an E. coli transcription block in the chloroplast, leading to an increase of foreign protein accumulation to up to 40% of TSP for Pal and 20% of TSP for Cpl-1. High dose-dependent bactericidal efficiency was shown for all three plant-derived lytic enzymes using their pathogenic target bacteria S. pyogenes and S. pneumoniae. Confirmation of specificity was obtained for the endotoxic proteins Pal and Cpl-1 by application to E. coli cultures. These results establish tobacco chloroplasts as a new cost-efficient and convenient production platform for phage lytic enzymes and address the greatest obstacle for clinical application. The present study is the first report of lysin production in a non-bacterial system. The properties of chloroplast-produced lysins described in this work, their stability, high accumulation rate and biological activity make them highly attractive candidates for future antibiotics. 2008 urn:nbn:de:kobv:517-opus-28950 Institut für Biochemie und Biologie