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

Abstract

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.