Transgenic parasites expressed PfEMP3-GFP or PfEMP3R>A-GFP chimeras from plasmids pPfEMP3WTGlux.1 or PfEMP3R>AGlux.1, respectively. Transgenic parasites expressing KAHRP-GFP contained the plasmid pKAHRPGlux.1, comprising DNA encoding residues 1-96 of KAHRP (PFB0100c). Parasites expressing ACPs-GFP are previously described 21 (link). Parasites expressing PfEMP3xQ-GFP contained the plasmid pPfEMP3xQGlux.1, encoding residues 1-36 of PfEMP3, followed by an alanine (to allow for signal peptidase cleavage at IYSEA-) and residues 63-82 of PfEMP3 (which include residues 63aQ64 of the PEXEL) cloned into pGlux.1. The plasmids pPMVHA1.5 pPMIXHA1.5 were integrated into either the Plasmepsin V locus (PF13_0133) or Plasmepsin IX locus (PF14_0281), respectively to append HA tags. The transgenic parasites 3D7-PMVmutHA episomally expressed plasmid pTETPMVmutHA, which contains DNA encoding Plasmepsin V with D118A, D365A and F370A substitutions fused to the same tags as in 3D7-PMVHA, cloned into the anhydrotetracycline (ATc) regulatable plasmid pTGFP 29 (link). The same DNA encoding Plasmepsin V with D118A, D365A and F370A substitutions fused to HA, but with alternate cloning sites, was cloned into pGlux.1, removing GFP, to create pPMVmutHA2. This plasmid contains the constitutively expressed crt promoter and is non-inducible. Recombinant GBP130 or GBP130 RLE>A was expressed in E. coli harbouring plasmids pGBP130-3Cmyc18His or pGBP130-3A-3Cmyc18His, respectively, which encodes residues 66 to 196 of GBP130 (containing the wild-type or mutant PEXEL). Recombinant Plasmepsin V was expressed in E. coli harbouring plasmid pHisPMVtrunc, which contains DNA encoding residues 37-521 of Plasmepsin V cloned into pProExHTb.
Plasmepsin V activity was assayed either with anti-HA beads containing ipPMVHA or ipPMVmutHA or 3–5 μg of HPLC-eluted protein added to the reaction that contained either PEXEL peptide substrate, recombinant PEXEL substrate or fluorogenic PEXEL peptide substrate. Digest reactions were analysed by HPLC and LC-MS/MS, immunoblot and fluorescence intensity, respectively.
Plasmepsin V activity was assayed either with anti-HA beads containing ipPMVHA or ipPMVmutHA or 3–5 μg of HPLC-eluted protein added to the reaction that contained either PEXEL peptide substrate, recombinant PEXEL substrate or fluorogenic PEXEL peptide substrate. Digest reactions were analysed by HPLC and LC-MS/MS, immunoblot and fluorescence intensity, respectively.
