Figure 3

Amount of soluble DsbA:HIV-1Pr and phosphate salts effect on chimeric protein production. A) Comparison by means of Western blot analysis of the amount of DsbA:HIV-1Pr protein expression in the soluble (S) and insoluble (I) cell fractions as obtained after disruption of BL21(DE3)pLysS E. coli cells (growth in TB medium, protein expression induced at the early-exponential phase) or of BL21-Codon Plus-(DE3)-RIL E. coli cells (protein expression induced at the early and middle-exponential phase of growth). B) Western blot analysis of DsbA:HIV-1Pr protein expression in total cell extracts of recombinant BL21-Codon Plus-(DE3)-RIL E. coli cells grown in the presence of a phosphate salts buffer system. LB: cells grown in LB medium (control); a: cells grown in LB medium added of 6.78 g/L of NaH₂PO₄ and 3 g/L of KH₂PO₄ (such as in M9 broth); b: cells grown in LB medium to which 9.4 g/L of K₂HPO₄ and 2.2 g/L of KH₂PO₄ were added (such as in TB broth). Protein expression was induced in all samples with 1 mM IPTG; cells were harvested at 1 or 3 hours after adding IPTG. An amount of cells corresponding to 0.2 mL of culture was loaded in each lane. C) Summary results of DsbA:HIV-1Pr production (see panel B): the expression level of cells grown on LB medium is reported as 100% (= 1.8 mg/L). Cells were collected at 1 hour (empty bars) and 3 hours (black bars) from IPTG addition. Western blot analysis was carried out by using anti-His-tag-specific antibodies. St: 0.5 μg of His-tagged recombinant D-amino acid oxidase.