Establishment of a comprehensive pathogen protein array for basic and applied research

Staphylococcus aureus represents a major gram-positive human pathogen showing an increasing resistance rate towards many latest-generation antibiotics. In light of this development, there is not only an urgent requirement to in-depth characterize basic S. aureus physiology, but also to accelerate the advancement and concomitant merging of high-throughput technologies focusing on innovative anti-staphylococcal therapy plus prevention strategies. Starting from our in-house established genome-wide collection of full-length S. aureus genes available in recombinational cloning compatible entry format, we thus propose to produce a systematic S. aureus expression ORFeome containing 2562 unique protein-encoding GST-tagged fusion constructs. This flexible resource creates the basis for subsequent innovative S. aureus whole-proteome chip fabrication. Although protein chips (arrays) have in general already been widely acknowledged for assessing biochemical activities or binding specificities in massively parallel fashion, numerous technical challenges related to array fabrication and limited content have, however, so far hampered their ubiquitous adoption. At this juncture, our meanwhile in part already optimized shotgun format in brief directly combines transformed E. coli colony spotting, protein expression, screening for interactors plus subsequent detection on a single nitrocellulose membrane. Applying a mixture of four different inducible E. coli expression strains - each with its own advantageous features -, the problem of bacterial inclusion body formation resulting in denatured protein conformation can be largely overcome. Therefore, the unbiased screening of such a comprehensive protein collection yields many unexpected associations that might otherwise be missed in a more limited design. Further substantial aim of our application will be to fabricate a bona fide focused protein array encompassing 351 recombinantly expressed and natively purified gene products selected for their essential participation in S. aureus growth and survival. Due to enclosed controls, both kinds of arrays will, despite differences in composition and quality, enable the standardization, signal-to-background quantification, quality estimation as well as inter-blot normalization of digitized images. We hence envision both products to play an important role in future S. aureus proteomics reasearch deciphering the molecular subnetworks governing medically relevant phenoma, plus in the discovery of diagnostic markers, vaccine candidates, and therapeutics.

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