Western blot analysis of the P80 variants. A. The P80 variants are represented by alignments of their proposed α helical regions and hydropathy profiles. B. After cultivating the different P80 clones for 2–5 h in LB-Amp medium proteins in the supernatant (after 5 h cultivation), the periplasmic fraction and the cell pellet (after 2 h cultivation) were separated by 9.5% SDS-PAGE and subjected to Western blot analysis using the P80 specific monoclonal antibody LF8. The samples correspond to 10 μl of the recombinant P80 (rP80) culture, 1 ml of P80Asn45Pro (N/P) culture, 10 μl of the helical P80 variant (Helic.) culture and 1 μl culture of the culture of the P80 variant without signal peptide (ΔSP). Samples from the culture supernatant were obtained from 30 μl (rP80), 0.8 ml (N/P), 0.2 ml (Helic.) and 60 μl (ΔSP) of media, and periplasmic proteins from 0.4 ml (rP80), 2 ml (N/P), 0.1 ml (Helic.) and 2 ml (ΔSP) of cultures. Additionally, lysate from 50 μl of culture and proteins from 250 μl of cell culture supernatant of M. hominis (strain FBG) were used. Marker, SeeBlue (Invitrogen, Germany).