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.

A New method for the capture of surface proteins inPlasmodium falciparumparasitized erythrocyte / Pantaleo, Antonella; Ferru, Emanuela; Turrini, Francesco Michelangelo. - 6:6(2012), pp. 536-541. [10.3855/jidc.2386]

A New method for the capture of surface proteins inPlasmodium falciparumparasitized erythrocyte

Pantaleo, Antonella;
2012-01-01

Abstract

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.
A New method for the capture of surface proteins inPlasmodium falciparumparasitized erythrocyte / Pantaleo, Antonella; Ferru, Emanuela; Turrini, Francesco Michelangelo. - 6:6(2012), pp. 536-541. [10.3855/jidc.2386]
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Utilizza questo identificativo per citare o creare un link a questo documento: https://hdl.handle.net/11388/262716
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