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Studien zur Optimierung der rekombinanten Genexpression in der methylotrophen Hefe Pichia pastoris

Studien zur Optimierung der rekombinanten Genexpression in der methylotrophen Hefe Pichia pastoris

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Prüfsumme: MD5:06ee55fea98cfec4ef729fa33a640b98

KABAOGLU, Fatma, 2005. Studien zur Optimierung der rekombinanten Genexpression in der methylotrophen Hefe Pichia pastoris [Dissertation]. Konstanz: University of Konstanz

@phdthesis{Kabaoglu2005Studi-7460, title={Studien zur Optimierung der rekombinanten Genexpression in der methylotrophen Hefe Pichia pastoris}, year={2005}, author={Kabaoglu, Fatma}, address={Konstanz}, school={Universität Konstanz} }

deu Kabaoglu, Fatma Studien zur Optimierung der rekombinanten Genexpression in der methylotrophen Hefe Pichia pastoris 2011-03-24T17:34:36Z application/pdf 2005 deposit-license 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. 2011-03-24T17:34:36Z Kabaoglu, Fatma Studies on the Optimization of Recombinant Gene Expression in Pichia pastoris

Dateiabrufe seit 01.10.2014 (Informationen über die Zugriffsstatistik)

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