Ferru, Emanuela and Pantaleo, Antonella and Turrini, Francesco Michelangelo (2012) A New method for the capture of surface proteins in Plasmodium falciparum parasitized erythrocyte. The Journal of Infection in Developing Countries, Vol. 6 (6), p. 536-541. eISSN 1972-2680. Article.
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Introduction: We propose a new method for the selective labeling, isolation and electrophoretic analysis of the Plasmodium falciparum protein exposed on the erythrocyte cell surface. Historically, membrane surface proteins have been isolated using a surface biotinylation followed by capture of biotin-conjugated protein via an avidin/streptavidin-coated solid support. The major drawback of the standard methods has been the labeling of internal proteins due to fast internalization of biotin.
Methodology: To solve this problem, we used a biotin label that does not permeate through the membrane. As a further precaution to avoid the purification of non surface exposed proteins, we directly challenged whole labeled cells with avidin coated beads and then solubilized them using non ionic detergents.
Results: A marked enrichment of most of the RBC membrane proteins known to face the external surface of the membrane validated the specificity of the method; furthermore, only small amounts of haemoglobin and cytoskeletal proteins were detected. A wide range of P. falciparum proteins were additionally described to be exposed on the erythrocyte surface. Some of them have been previously observed and used as vaccine candidates while a number of newly described antigens have been presently identified. Those antigens require further characterization and validation with additional methods.
Conclusion: Surface proteins preparations were very reproducible and identification of proteins by mass spectrometry has been demonstrated to be feasible and effective.
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