Figure 4. The L1-2 loop of TssJ is required for TssJ-TssM complex formation.

(A) In vivo Hcp release assay. Hcp_FLAG release was assessed by separating whole cells (WC) and supernatant (Sn) fractions from tssJ cells carrying the empty vector (tssJ), the vector encoding wild-type TssJ (tssJ^WT) or the vector encoding the TssJ-ΔL1-2 mutant (tssJ^ΔL1-2). 2 ×10⁸ cells and the TCA-precipitated material of the supernatant from 5×10⁸ cells were loaded on a 12.5%-acrylamide SDS-PAGE and immunodetected using the anti-FLAG monoclonal antibody (lower panel) and the anti-TolB polyclonal antibodies (lysis control; upper panel). (B) Solubilized extracts of E. coli K12 W3110 strain producing (+) or not (-) FLAG-tagged TssM-ekto and HA-tagged TssJ or TssJ-ΔL1-2 mutant were subjected to immunoprecipitation with anti-FLAG-coupled beads. The total solubilized material (T) and the immunoprecipitated material (IP) were loaded on a 12.5%-acrylamide SDS PAGE, and immunodetected with anti-HA (TssJ and TssJ-ΔL1-2; lower panel) and anti-FLAG (TssM-ekto; upper panel) monoclonal antibodies. Immunodetected proteins are indicated on the right. Molecular weight markers are indicated on the left. (C) Affinity purification of TssJ-ΔL1-2 with TRX-His6-TssM-ekto. The Coomassie blue-stained SDS-PAGE shows the fractions of the purification steps (Load, fraction 1; Wash, fractions 2-4; Elution, fractions 5 and 6). The positions of the proteins of interest are indicated on the right.