Studien zur Optimierung der rekombinanten Genexpression in der methylotrophen Hefe Pichia pastoris
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Zusammenfassung
The methylotrophic yeast Pichia pastoris was used as a possible eukaryotic expression and secretion system for the evaluation of a functional protein tech-nology (FunProTec). Human phosphodiesterase 1B1 and 10A, a truncated form of human lung surfactant protein D (SP-D(N/CRD)), lipase A from Bacillus subtilis 168M and alkaline phosphatase from E. coli K-12 were chosen as examples for heterologous gene expression. Gene expression and protein secretion was ob-served for all proteins except for phosphodiesterase 10A.
The analysis of the expression products of SP-D(N/CRD) showed that the recom-binant protein was proteolytically degraded. Several experiments, including physiological and molecular approaches, were performed to avoid proteolytic deg-radation of recombinant SP-D(N/CRD). Only when the inhibitor of aspartic pro-teinases pepstatin (15 µg/mL) was added to the medium a significant decrease of protein degradation could be observed. This experiment also showed that proba-bly al least one aspartic proteinase was involved in the proteolytic degradation of SP-D(N/CRD).
Based on the preliminary P. pastoris genome sequence data proteinase A and seven yet undescribed aspartic proteinases destinated aspartic proteinase 1 to aspartic proteinase 7 were identified. The sequence data of these proteinases were verified by cloning experiments and subjected to bioinformatic studies so that conclusions concerning the primary structure of the proteinases were drawn.
Protease-deficient strains of aspartic proteinase 3 and aspartic proteinase 5 were constructed in P. pastoris X-33. Expression of recombinant SP-D(N/CRD) was carried out in the designed protease-deficient strains and showed to be different in comparison to the wildtype strain.
Zusammenfassung in einer weiteren Sprache
Die methylotrophe Hefe Pichia pastoris wurde als eukaryotisches Expressions- und Sekretionssystem zur Evaluierung einer Funktionellen Protein Technologie (FunProTec) getestet. Die humanen Phosphodiesterasen 10A und 1B1 (kPDE10A, PDE1B1), das humane Lungensurfactant Protein D (SP-D(N/CRD)), die alkalische Phosphatase (PhoA) aus E. coli K-12 und die Lipase A (LipA) aus B. subtilis 168M wurden als Beispielproteine für die heterologe Genexpression gewählt. Außer für kPDE10A wurde die Expression und Sekretion aller Proteine in das Medium gezeigt.
Bei der Expression von rekombinantem SP-D(N/CRD) wurde gezeigt, dass die Expressionsprodukte proteolytisch degradiert waren. Es wurden mehrere physio-logische und molekularbiologische Strategien zur Minimierung der Protein-degradation von rekombinantem SP-D(N/CRD) verfolgt. Durch den Einsatz von Pepstatin (15 µg/mL) wurde bei der Schüttelkolbenexpression ein Rückgang der Degradation von SP-D(N/CRD) erzielt. Dieses Experiment zeigte auch, dass mög-licherweise eine Aspartat-Peptidase an der Degradation von SP-D(N/CRD) betei-ligt war.
Ausgehend von der P. pastoris Genomsequenz wurden acht Aspartat-Peptidasen identifiziert: Proteinase A und sieben weitere, vorher nicht beschriebene, Aspartat-Peptidasen (Aspartat-Peptidase 1 bis Aspartat-Peptidase 7). Diese neu identifizierten Aspartat-Peptidasen wurden kloniert und mittels bioinformatischer Studien untersucht. Es konnten mögliche Aussagen über wichtige Strukturelemente getroffen werden.
Die Aspartat-Peptidasen 3 und 5 wurden in dem P. pastoris-Stamm X-33 durch homologe Rekombination deletiert. Die Expression von rekombinantem SP-D(N/CRD) erfolgte in beiden Protease-defizienten Stämmen und zeigte vor allem bei dem Stamm P. pastoris X-33deltaAP-5-SP-D(N/CRD) ein verändertes Degradationsprofil im Vergleich zum Wildtyp-Stamm.
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KABAOGLU, Fatma, 2005. Studien zur Optimierung der rekombinanten Genexpression in der methylotrophen Hefe Pichia pastoris [Dissertation]. Konstanz: University of KonstanzBibTex
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<dcterms:abstract xml:lang="eng">The methylotrophic yeast Pichia pastoris was used as a possible eukaryotic expression and secretion system for the evaluation of a functional protein tech-nology (FunProTec). Human phosphodiesterase 1B1 and 10A, a truncated form of human lung surfactant protein D (SP-D(N/CRD)), lipase A from Bacillus subtilis 168M and alkaline phosphatase from E. coli K-12 were chosen as examples for heterologous gene expression. Gene expression and protein secretion was ob-served for all proteins except for phosphodiesterase 10A.<br /><br />The analysis of the expression products of SP-D(N/CRD) showed that the recom-binant protein was proteolytically degraded. Several experiments, including physiological and molecular approaches, were performed to avoid proteolytic deg-radation of recombinant SP-D(N/CRD). Only when the inhibitor of aspartic pro-teinases pepstatin (15 µg/mL) was added to the medium a significant decrease of protein degradation could be observed. This experiment also showed that proba-bly al least one aspartic proteinase was involved in the proteolytic degradation of SP-D(N/CRD).<br /><br />Based on the preliminary P. pastoris genome sequence data proteinase A and seven yet undescribed aspartic proteinases destinated aspartic proteinase 1 to aspartic proteinase 7 were identified. The sequence data of these proteinases were verified by cloning experiments and subjected to bioinformatic studies so that conclusions concerning the primary structure of the proteinases were drawn.<br /><br />Protease-deficient strains of aspartic proteinase 3 and aspartic proteinase 5 were constructed in P. pastoris X-33. Expression of recombinant SP-D(N/CRD) was carried out in the designed protease-deficient strains and showed to be different in comparison to the wildtype strain.</dcterms:abstract>
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