We found three known Fn/Fg-binding polypeptides (ΔFnBPA, ΔEbh and ΔCoa) and in addition five polypeptides of novel adhesive function (ΔPurK, ΔUsp, ΔSCOR, ΔIspD and ΔPBP). The cloned chromosomal fragments frequently encoded polypeptides below the AZD2171 purchase length of intact binding domains of large staphylococcal adhesins, such as the clumping factors (ClfA, ClfB) and SD-rich fibrinogen-binding proteins [14, 42]. Hence, in future applications of the presented technique longer chromosomal fragments should preferentially be cloned. We did however identify several fibronectin-binding
polypeptides, which possibly is explained by the EPZ015666 mouse fact that short fragments of typical fibronectin-binding MSCRAMMs mediate high-affinity binding [43]. The observed variation in concentration of FLAG-tagged polypeptides in the cell-free supernatants of the Ftp-library clones, which was due to variable expression of the cloned S. aureus chromosomal fragments in E. coli and may have an effect on the screening results, could be circumvented by quantification of the polypeptides prior to the analysis. The findings obtained by primary screening of Ftp library clones were confirmed by ELISA and SPR analyses using corresponding purified His-tagged recombinant polypeptides. All the binding results are combined Elafibranor research buy in Table
3, and strongly indicate that the Fn- and Fg-binding polypeptides ΔFnBPA, ΔPurK, ΔCoa, ΔUsp and ΔEbh truly Teicoplanin have adhesive functions under the tested conditions. The very weak interactions observed with ΔPBP (with Fn and Fg) and ΔUsp (with CIV) require further verifications and could not be confirmed by ELISA or SPR using 6xHis polypeptides. Some discrepancies were observed with
the Ebh polypeptides, which may be due to the protein itself or the methods applied for verification of the results. In the ELISA assays, ΔEbh and His-ΔEbh bound to Fn, whereas interaction with Fn as well as Fg was observed in the SPR analysis. Fg is not known to be a ligand for Ebh in the literature, but Ebh is a giant protein, 9535 amino acid residues in length [34], and may have unknown properties. ELISA is an end-point type of analysis, whereas SPR is a real-time analysis considered to be very sensitive and optimal for detection of weak interactions [44]. Thus, the SPR technology may in this case have revealed a novel function of Ebh, which remains to be further characterized in a coming study. The verification of the interactions of ΔSCOR and ΔIspD (with Fn and Fg) was hampered since the polypeptides could not be produced as purified His-tagged polypeptides by conventional expression technology. Table 3 A summary of the binding of S.
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