Herstellung einer Zinkfinger-Sonde zum Einsatz in DNA-basierter Staphylococcus aureus-Diagnostik

Staphylococcus aureus, especially Methicillin?resistant S. aureus (MRSA), is the leading cause of nosocomial and communal infections worldwide. The dection using standard cultivation techniques is currently time and cost consuming, whereas nucleic acid based diagnostics offer rapid outcomes, high sensitivity and specificity, multiplex applications and information about antibiotic susceptibility. The recognition of native DNA under physiological conditions without required denaturation steps are feasible using zinc finger proteins. Thus, the investigation was focussed on the establishment of an artificial hybrid protein containing the zinc finger domain (ZFD) of the well characterized human transcription factor Sp1 and Sp3, respectively. Both bind specifically to the GC-box (5`-GGGGCGGGG-3´), present in the genome of S. aureus. The ZFD of Sp3 and Sp1 were amplified by PCR and cloned into the eukaryotic expression system pEGFP?C1 and into the prokaryotic expression system pMAL-c2X, respectively. The eukaryotic protein expression of EGFP-Sp3-ZFD in HEK293 cells was ineffective due to proteasomal degradation indicated by the stabilization of EGFP-Sp3-ZFD after proteasome inhibitor treatment. However, the prokaryotic protein MBP-Sp1-ZFD could be successfully overexpressed in E. coli BL21(DE3) and purified using amylose affinity chromatography, which is confirmed by SDS-PAGE and Western Blot. The functionality of MBP-Sp1-ZFD was determined by Electrophoretic Mobility Shift Assay (EMSA) experiments showing the formation of complexes with specific dsDNA, which could be deminished in a concentration dependent manner using EDTA. Unspecific dsDNA of two, three or eight base pair substitutions in the sequence of the GC-box, were bound less. Analysis of Surface Plasmon Resonance (SPR) measurements reveals comparable results. The findings showed dissociation constants according to data achieved for the native transcription factor Sp1. Significant differences in the association constant ka, specific dsDNA of 10 Ms 11 1 1and unspecific dsDNA as well as specific ssDNA of 10 Ms, 6-1-1were observed. The results indicated clearly, that the ZFD of MBP-Sp1-ZFD was responsible for the interaction with DNA. Thus, ZF could be utilized as diagnostic tool in multiplexed assays by using sensitive biosensors, e.g. SPR, to discriminate between specific and unspecific dsDNA.