Additional File 1:

ClustalW alignment of PfM18AAP with Homo sapiens, Saccharomyces cerevisiae, and other Plasmodium homologues. The sequences, H. sapiens (Hs) (Q9ULAO), S. cerevisiae (Sc) (P38821), P. falciparum (Pf) (PFI1570c), P. chabaudi chabaudi (Pc) (PC000238.00.0), P. yoelii yoelii (Py) (PY03205), P. knowlesi (Pk) (PKH_073050), and P. vivax (Pv) (Pv087090)) were aligned using the ClustalW program. The five amino acids (blue) that bind the co-factor and the two amino acids (red) that cleave the substrate are conserved amongst all the species. An additional histidine (yellow) involved in enzymatic activity and another histidine (green) involved in quaternary structure stabilization are also marked. The 33 amino acid spectrin-binding region (orange) is only present in the P. falciparum aspartyl aminopeptidase.

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Additional File 2:

Ferguson plot and molecular weight standard curve used to determine the approximate molecular weight of the rPfM18AAP subunits visualized on the non-denaturing agarose/polyacrylamide gels (figure 2). Ferguson plot (left) showing the log R_m of the rPfM18AAP oligomeric forms at different polyacrylamide percentages and double-log graph (right) of the negative slopes (obtained from Ferguson plots) versus the molecular weight of each standard (spectrin and BSA multimers). Only three rPfM18AAP subunits (monomer, faint dimer and tetramer) were distinctly visible on the non-denaturing agarose/polyacrylamide gels. The higher oligomers separated as a smear (figure 2). The Ferguson plot (left) shows that rPfM18AAP subunits are oligomers of each other because the lines intersect at ~3% gel concentration. Ferguson plot symbols: squares – rPfM18AAP monomer; triangles – rPfM18AAP dimer; crosses – rPfM18AAP tetramer. The molecular weight of the three rPfM18AAP oligomeric forms was determined from the standard curve (right) as ~70 kDa (monomer), ~155 kDa (dimer), and ~290 kDa (tetramer). Standard curve crosses: yellow – BSA (66 and 132 kDa); blue – spectrin (dimer, 460 kDa; tetramer, 920 kDa; and hexamer, 1380 kDa); red – rPfM18AAP.

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Additional File 3:

Blot overlay assay performed in the absence of rPfM18AAP. Laemmli SDS-polyacrylamide gel (left) and blot overlay (right) showing that the PentaHis™ HRP Conjugate antibody does not bind to BSA or any of the erythrocyte membrane proteins when the assay is performed without rPfM18AAP. Lane 1 – bovine serum albumin; lane 2 – rPfM18AAP (positive control); lane 3 – erythrocyte membrane proteins.

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