Fig. 5.

Substitution of the MSP1 secondary processing site with a PfSUB1-sensitive sequence.A. Western blot time-course analysis of rPfSUB1-mediated processing of rMSP1_38/42 either in wild-type form (WT) or after modification of the secondary processing site by site-directed mutagenesis to convert it to a predicted PfSUB1-sensitive site (SUB2to1). The WT rMSP1_38/42 (80 kDa, arrowed) is converted to a stable 42 kDa terminal species (open round-head lollipop) equivalent to MSP1₄₂. The SUB2to1 mutant (arrowed) is similarly converted to the normal MSP1₄₂ species (open round-head lollipop), but also undergoes an additional processing step that converts it via an approximate 70 kDa intermediate (closed square-head lollipop) to a stable 33 kDa form (open square-head lollipop). The 2 h tracks show the results of incubation for 2 h in the absence of added rPfSUB1. The bands marked with a star are likely truncated forms of rMSP1_38/42 derived from breakdown of the recombinant protein during its purification and refolding.B and C. Schematic depiction of processing patterns for the WT (B) and SUB2to1 mutant rMSP1_38/42 protein (C). The relative position of the secondary processing site, the sequence modifications made and the region of the protein recognized by the rabbit anti-MSP1₄₂ antibodies used to probe the blots, are indicated. Individual processed species are marked with arrow or lollipop symbols to aid comparison with the corresponding bands on the blots.