The unsuccessful attempts to truncate the N-terminal portion of EXP1 indicate an essential function of this region during asexual blood stage growth.

Exp1 (HEP17) is a protein that localizes to the parasitophorous vacuole membrane (PVM) of blood stages and liver stages
Pb/PfEXP1’s topology includes an N-terminal (NT) domain (aa 1-75/1-79) that harbors a classical signal peptide (aa 1-23); a putative catalytic domain that includes the Pf Arg70 residue and is presumed to be responsible for GST activity in blood stage parasites; an internal hydrophobic region typical of transmembrane anchor sequences (aa 75-97/79-101); and a C-terminal (CT) domain (aa 97-166/101-162) that includes a C1 (aa 103-136/107-134) and a C2 (aa 137-166/135-162) region, as defined in (Sa et al., 2017). In both blood and liver parasite stages, EXP1 is proposed to be integrated into the PVM with the N-terminus facing the lumen of the PV, and the C-terminus exposed to the host cell cytosol.

In order to dissect the functional role of different domains of PbEXP1, we attempted to generate modified versions of this protein bearing a truncated version of its N- and C-terminal domains. Despite multiple attempts, we were unable to generate a modified version of PbEXP1 lacking the NT domain (PbEXP1ΔNT; RMgm-4656), suggesting that it is essential for blood stage development of Pb parasites. However, transgenic Pb parasite lines expressing a CT domain-truncated version of PbEXP1 were successfully generated in both the PbANKA (PbWT) and PbGFPcon (PbGFP) parental parasite lines, yielding their PbEXP1ΔCT and PbGFPEXP1ΔCT mutated counterparts, respectively, and showing that Pb blood stages can develop in the absence of the PbEXP1 CT domain. Successful deletion of the CT domain in the PbEXP1ΔCT parasites was confirmed by Western blot using two different anti-EXP1 antibodies that bind either to epitopes present on the full length PbEXP1 protein or to epitopes present only on the protein’s C-terminus.