PMC

Dual pathways for disulfide bond reduction in the cytoplasm of E. coli. Dual pathways for disulfide bond reduction in the cytoplasm of E. coli. On the knockout of both pathways the reduction of disulfide bonds is inhibited, but there is no active system to catalyze their formation.

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.

Production of PhoA in the cytoplasm of E. coli. A) SDS-PAGE analysis of the production of PhoA produced in LB media at 30°C. T = total E. coli lysate, S = E. coli lysate soluble fraction, P = purified protein from BL21 with co-expression of Erv1p from the NEM-treated lysate and is representative of the quality obtained from all of the samples. Mw = molecular weight markers. BL = BL21 (DE3) pLysSRARE; RG = rosetta-gami; + E = co-expression of S. cerevisiae Erv1p from a polycistronic vector. The positions of PhoA (upper) and Erv1p (lower) are marked with arrows. B) Relative yields of active PhoA normalized to the system producing the most active protein and shown as percentage mean ± s.d. (n = 4). C) Representative blot from a shift-assay based on alkylation of free thiol groups to examine the disulfide bond status of the PhoA produced. Note that the samples are treated with the thiol-blocking agent N-ethylmaleimide (NEM) before reduction and maleimide based addition of polyethyleneglycol. Hence an increase in apparent molecular weight is consistent with the presence of one or more disulfide bonds in the original sample. The greater the number of disulfide bonds the greater the mass shift. PhoA produced in both the Δgor ΔtrxB background and in the wild-type background plus co-expression of Erv1p show a homogeneous disulfide bonded protein being produced, however this assay does not determine whether these disulfide bonds are native or not. D) Specific activity (μmole of product formed per minute per mg of protein) of purified PhoA shown as mean ± s.d. (n = 3).