Figure 4
Comparative analysis of the solubilization capacity and immunogenicity of PfTrx and EcTrx.
(a) The 1-120 aa. N-terminal region of L2 inserted into the display site of either EcTrx or PfTrx was used to test the solubilisation capacity of the two scaffold proteins. Bacterial cells expressing the two His-tagged Trx-L2(1-120) proteins (marked with an arrow) were cultured in parallel (10 ml culture each), transferred to lysis buffer (1 ml) and the resulting lysate (“induced culture”, I) was centrifuged in order to separate pellet-associated, insoluble proteins (P) from soluble proteins recovered in the supernatant (S). An equal volume of the various fractions (15 µl each), including total lysates derived from uninduced bacterial cultures (U), was subjected to SDS-PAGE fractionation. A sizeable fraction (>50%) of the PfTrx-L2(1-120) polypeptide was present in the soluble fraction, whereas EcTrx-L2(1-120) was almost completely insoluble (cf. lanes 3 and 4 with lanes 7 and 8). The results of an immunoblot analysis performed with an anti-L2(20-38) monoclonal antibody, using scaled-down amounts of the various fractions (0.08 µl equivalents each), are shown below the Coomassie-stained gel image. (b) HPV16 neutralization titers measured in sera collected after the last immunization (3rd boost) from mice immunized with either affinity-purified EcTrx-L2(20-38)3 or PfTrx-L2(20-38)3 adjuvanted with 50% v/v Montanide ISA720. Dots represent individual neutralization titers, i.e., the maximum dilution of immune sera from the two groups (10 animals each) causing 50% pseudovirion neutralization; geometric means of the titers for each group are indicated by horizontal lines (see ‘Methods’ for further details).
