Fig. 4

Mapping the regions in PrsA that interact with SpA and the formation of the PrsA dimer. (A) Map of PrsA and its truncated mutants. Based on the sequence homology with the B. subtilis PrsA protein, PrsA from S. aureus HG001 contains a signal peptide (SP), an N-terminal, a PPIase, and a C-terminal domains. (B) PrsA and its truncated mutants were expressed in E. coli BL21(DE3). Protein A-agarose beads were added and mixed with the cell lysates. Proteins binding to the beads were eluted and detected by immunoblotting using anti-PrsA antibody. (C) E. coli BL21(DE3)(pET-PrsA) (FL), E. coli BL21(DE3)(pET-PrsA-D3) (D3) and E. coli BL21(DE3)(pET-PrsA-D4) (D4) were treated with 1% formaldehyde. His-tag proteins were purified from E. coli using Ni-NTA beads. Proteins solubilized in sample buffer were incubated at 37 °C for 10 min (lanes 1, 3, 5, 7, 9, 11) or at 95 °C for 30 min (lanes 2, 4, 6, 8, 10, 12) and then separated by SDSȁPAGE, followed with Coomassie blue staining (CBS, lanes 1, 2, 5, 6, 9, 10). PrsA in the protein samples was determined by immunoblotting (IB, lanes 3, 4, 7, 8, 11, 12) using anti-PrsA antibody. Protein bands within boxes were cut and analyzed by LC MS/MS