Rekombinante Produktion, Aufreinigung und Kristallisation der Shigella Pathogenitätsfaktoren Spa15, IpgC und OspD1

Many gram-negative bacteria, including Shigella flexneri, use a type III secretion apparatus (T3SA) serving the secretion of bacterial proteins into eukaryotic cells. These proteins enable bacteria to infect eukaryotic cells. About 20 structural proteins and chaperones encoded by the mxi-spa-operon are needed to form the T3SA. The so-called "early genes" of the Shigella virulence plasmid obtain information for the synthesis of the structural proteins of the T3SA and invasins. They are regulated by VirF. The "later read-off genes" of the plasmid ensure that the host cell remains alive and are transcriptionally regulated by MxiE. Their transcriptional activation is achieved by activating the T3SA. The main focus of this thesis lies on the pathogenicity factor OspD1. The interaction of OspD1 with the chaperone Spa 15 and the transcriptional regulator MxiE was investigated biochemically and by X-ray crystallography. Within this thesis, an improved expression system was established which enables the purification via the maltose-binding protein-tag and finally results in pure OspD1 protein. After extensive attempts crystallization conditions (at 4 °C) were found which enable crystal growth of OspD1. The following improvement of the crystallization condition leads to a native data set of 2.34 Å. Unfortunately, the structure of OspD1 could not be solved within the scope of this thesis. Attempts to solve the structure by molecular replacement were not successful due to the absence of a suitable structural model with sufficient homology. Subsequent extensive efforts using heavy metal derivatives resulted in several datasets, which however, could not be evaluated. The constructed selenomethionine derivative, which in general enables structure elucidation using multi-wavelength anomalous diffraction (MAD), was expressed successfully but could not be purified. In order to crystallographically characterize the interaction of Spa15 with OspD1, co-crystallization experiments of the complex as well as crystallisation attempts of Spa15 with synthesized OspD1peptides of different length were conducted. The quantitative studies of protein interactions between Spa15 and OspD1 were performed by microscale thermophoresis. This relatively new method is based on the directed movement of molecules along a temperature gradient, which is influenced in a measurable and concentration-dependent manner by the binding of a ligand. For interaction of Spa15 with OspD1 a KD of about 40 nM was determined. Thereafter, various OspD1 peptides were measured with Spa15 to investigate the binding of individual peptides and determine the loss of the binding strength compared to the full-length protein. Furthermore, this method was used to investigate the binding of the MxiE peptides. In addition, the binding of MxiE and IpaC peptides to the chaperone IpgC was tested by microscale thermophoresis and co-crystallization experiments were conducted with the most potent peptides. Within this thesis, it was possible to find and improve crystallization conditions which results in a dataset with a resolution of 2.6 Å measured at BESSY in Berlin. However, no peptide was found in the refined crystal structure. For the biochemical characterisation of IpgC, the fragment library of the AG Klebe, consisting of over 320 fragments, was screened against IpgC utilizing the thermal shift assay. Within this project, it was investigated whether individual fragments have the potential to bind to IpgC.