Untersuchungen zur Struktur und Funktion der Transportdomänen des Autodisplay Systems (English shop)

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Autodisplay provides a system that enables the presentation of a functional passenger protein on the surface of E. coli cells. In this work, the carboxy-terminal amino acid (phenylalanine) of the ß-barrel domain of the Autodisplay system was exchanged. By means of site-directed mutagenesis it was replaced by tryptophan, tyrosine, histidine or valine, or deleted entirely. Aromatic amino acids could be exchanged for one another without loss of function. Investigations on the valine mutant (hydrophobic but not aromatic) demonstrated that integration or transport, but not the conformation of the protein, was impaired. When the C-terminal amino acid was deleted, no protein located in the outer membrane could be detected. Thus, a direct influence of the aromaticity of the C-terminus on integration into the outer membrane could be demonstrated. Over the course of time, evolution and selection led to seven amino acid exchanges in the transport domains. The mutations had no influence on the properties of Autodisplay examined in this work, such as the amount of surface-displayed passenger. Overexpression gave rise to two different protein forms. Only one form showed the heat denaturation behaviour typical of ß-barrel proteins and displayed its passenger domain on the surface. The other form was in a non-functional conformation and did not transport the passenger across the outer membrane. Analysis of the N-terminal protein sequence demonstrated that only the functional protein had been processed by signal peptidase. Solubilisation in the detergent sodium N-lauroyl sarcosinate made it possible to separate the two forms from each other. The functional protein behaved exactly like the natural outer membrane protein OmpA, which served as an indicator protein and likewise forms a ß-barrel. Protein expression could be optimised by changing the induction conditions to such an extent that only the functional variant was formed, without impairing the folding of the structure or the number of surface-displayed passenger molecules. Co-expression of the periplasmic chaperones SurA, FkpA, DsbA and DsbC made it possible to double the amount of surface-displayed protein.