Figure 6
Production and properties of the tag-free, heat-purified form of the PfTrx-L2(20-38)3 antigen.
(a) SDS-PAGE analysis of PfTrx-L2(20-38)3 without His-tag purified with the one-step thermal purification procedure (see ‘Methods’ for details). I: induced bacterial culture; U: uninduced culture; P: post-lysis pellet; S: post-lysis supernatant; PT: heat-treated post-lysis pellet; ST: heat-treated post-lysis supernatant, containing the purified (>90%) PfTrx-L2(20-38)3 protein (marked with an arrow). (b) Far-UV CD analysis of affinity-purified EcTrx-L2(20-38)3 (left) and PfTrx-L2(20-38)3 (right) subjected to the same heat-treatment utilized for thermal purification. Spectra of the unheated proteins (25°C), of the proteins incubated under thermal purification conditions (20 min at 70°C) and of the latter samples returned to 25°C are shown as solid, dotted and dashed lines, respectively. (c) Limited proteolysis analysis. Heat-purified EcTrx-L2(20-38)3 and PfTrx-L2(20-38)3 were treated for the indicated times with chymotrypsin (enzyme:Trx antigen ratio of 1:100 w/w), fractionated by SDS-PAGE and either stained with Coomassie Blue R-250 (leftpanel) or subjected to immunoblot analysis with an anti-L2(20-38) mAb (right panel). A sample of each heat-purified Trx-L2(20-38)3 protein not treated with chymotrypsin (Nt) served as control for these experiments (see ‘Methods’ for details). (d) HPV16 neutralization capacity of sera collected after the last immunization from mice immunized with the metal affinity-purified or the heat-purified PfTrx-L2(20-38)3 antigen adjuvanted with 50% v/v Montanide ISA720. Dots represent neutralization titers measured in sera from individual immunized animals; geometric means of the titers for the two groups (8 animals/each) are indicated by horizontal lines (see legend of Fig. 4b and ‘Methods’ for further details).
