Figure 2
Expression analysis of chimeric RTB/p24 and p24 in E. coli. Total proteins were extracted from E. coli cultures, separated by 10% SDS-PAGE and stained with Coomassie. Protein profiles were analyzed after 5 h induction with (5 h+) or without IPTG (5 h-) and compared with non-induced cultures (T0). Four different E. coli strains were tested and these are indicated in the lower part of the panels. Panel A, p24 construct. The arrow indicates the expected band of about 34 kDa. Panels B and C, RTB/p24 construct. The arrows indicate the expected chimeric protein (RTB/p24) of about 57 kDa. The six E. coli strains employed are indicated at the bottom of panels. Panel D, western blot of RTB/p24 after expression at two temperatures (21°C and 37°C). We analyzed supernatant (S) and pellet (P) fractions for determination of soluble and insoluble fractions in E. coli cultures after 5 h induction. T0 represents non-induced cultures. Western blot was performed using anti-His monoclonal antibody at dilution of 1/1000. Panel E, analysis of different pH and NaCl concentrations on the lysis buffer to improve the solubility of RTB/p24 chimeric protein during affinity chromatography. Lanes 1 and 2 represent 1 ml aliquots of collected fractions. The arrows indicate the expected chimeric protein (RTB/p24). In all panels, M represents molecular weight markers in kDa.