An EGF-like Protein Forms a Complex with PfRh5 and Is Required for Invasion of Human Erythrocytes by Plasmodium falciparum
Figure 1
The processed 45 kDa PfRh5 C-terminal domain exists as a larger complex.
(A) PfRh5 partially purified from parasite culture supernatant was analysed by size exclusion chromatography on a Superdex 200 analytical column. PfRh5 was eluted from the column as a ∼150–200 kDa species. Molecular weight of eluted protein fractions are indicated by arrows as shown using standard proteins of known size. PfRh5 was detected using 2F1, a specific monoclonal antibody to the protein. (B) Blue native gel electrophoresis confirmed that PfRh5 migrates as a ∼150–200 kDa species. The processed PfRh1 fragment of 110 kDa is included as a control. (C) 300 µl of the PfRh5-containing fraction, isolated from culture supernatant, was loaded onto a Superdex 200 analytical column and eluted with PBS. (D) An identical 300 µl sample was pre-incubated with 25 µg of monoclonal anti-PfRh5 antibody before loading onto the same Superdex 200 column. The peak of the eluted PfRh5 protein lies at two preceding fractions as compared to panel C, corresponding to an increase in molecular weight of ∼150 kDa indicating that one antibody molecule was bound to the PfRh5-containing species. The monoclonal antibody-bound PfRh5 is indicated by an arrow. PfRh5 and the immunoglobulin heavy and light chain of antibody seen in fraction #22–25 are the free PfRh5 complex and the excess antibody. The asterisks refer to cross-hybridising bands corresponding to the immunoglobulin heavy and light chain.