fig1

Figure 1. Effect of DnaJ on the co-translational solubilization and membrane insertion of MS2-L. All expressions were carried out in BW25113ΔDnaJ lysate (A) MS2-L was P-CF expressed in the presence of increasing DnaJ concentrations (0, 10, 30, and 100 µM). Solubilization efficiencies were determined by densitometry from immunoblots of pellet and supernatant fractions. Combined signals of pellet and supernatant were normalized to 1. Error bars represent the SEM of n = 6 (n = 5 for 100 µM DnaJ) biological replicates; (B) MS2-L was either P-CF or L-CF (10 µM DMPG NDs) synthesized in the presence or absence of 100 µM DnaJ. Solubilized MS2-L was quantified as described in (A). Error bars indicate the SEM of n = 6 (n = 5 for P-CF + 100 µM DnaJ) biological replicates; (C) Complex formation of DnaJ with D-CF synthesized MS2-L. After expression in the presence of Brij78 and 30 µM DnaJ, the toxin was StrepII-purified from the RM. As verified by SDS-PAGE (left panel) and immunoblot (right panel), the chaperone (containing a His₆-tag) co-purified with MS2-L. 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; RM: reaction mix.