Figure 3

Production of AppA in the cytoplasm of E. coli. A) Relative yields of active AppA produced in LB media at 30°C, normalized to the system producing the most active protein and shown as percentage mean ± s.d. (n = 4). BL = BL21 (DE3) pLysSRARE; RG = rosetta-gami; + = co-expression from a polycistronic vector where D = mature E. coli DsbC, E = S. cerevisiae Erv1p. B) Representative blot from a shift-assay based on alkylation of free thiol groups to examine the disulfide bond status of the AppA produced. While AppA produced upon co-expression of Erv1p in a wild-type background shows a homogeneous disulfide bonded protein being produced, the protein produced in the Δgor ΔtrxB background shows heterogeneity and a lower degree of disulfide bond formation. Note that the molecular weight of the mal-PEG is not homogenous and hence modified proteins, especially those with multiple mal-PEG added, appear as more defuse bands.