fig2

Figure 2. Post-translational interaction of DnaJ with MS2-L derivatives in detergent and in NDs. Toxin expressions were carried out in the BW25113ΔDnaJ lysate. MS2-L derivatives were expressed and solubilized co-translationally in either Brij78 (D-CF) or DMPG NDs (L-CF). Purified toxins were then analyzed in pulldown experiments for interaction with DnaJ. The MS2-L derivatives were immobilized via their C-terminal StrepII-tag and 50 µg purified DnaJ was added as prey. After washing, the formed complexes were eluted in 40 µL of SDS-loading buffer and 15 µL samples were analyzed by SDS-PAGE. Bound DnaJ was detected by immunoblotting with α-His antibodies and immobilized MS2-L derivatives were visualized with α-StrepII antibodies. (1) MS2-L; (2) MS2-Lp₂₃; (3) MS2-Lp₂₆; (4) MS2-Lp₂₉; (5) MS2-Lp₃₂; (6) MS2-Lp₃₅. (A) D-CF expression in Brij78; (B) L-CF expression in DMPG NDs. The smaller band detected in the DnaJ control is a putative degradation product. D-CF: Detergent-based cell-free expression; DMPG: 1,2-dimyristoyl-sn-glycero-3-phospho-(1’-rac-glycerol); ND: nanodisc; P-CF: precipitate forming cell-free expression.