Article: article from journal or magazin.
Expression and one-step purification of Plasmodium proteins in dictyostelium.
Molecular and Biochemical Parasitology
Nearly full-length Circumsporozoite protein (CSP) from Plasmodium falciparum, the C-terminal fragments from both P. falciparm and P. yoelii CSP and a fragment comprising 351 amino acids of P.vivax MSPI were expressed in the slime mold Dictyostelium discoideum. Discoidin-tag expression vectors allowed both high yields of these proteins and their purification by a nearly single-step procedure. We exploited the galactose binding activity of Discoidin Ia to separate the fusion proteins by affinity chromatography on Sepharose-4B columns. Inclusion of a thrombin recognition site allowed cleavage of the Discoidin-tag from the fusion protein. Partial secretion of the protein was obtained via an ER independent pathway, whereas routing the recombinant proteins to the ER resulted in glycosylation and retention. Yields of proteins ranged from 0.08 to 3 mg l(-1) depending on the protein sequence and the purification conditions. The recognition of purified MSPI by sera from P. vivax malaria patients was used to confirm the native conformation of the protein expressed in Dictyostelium. The simple purification procedure described here, based on Sepharose-4B, should facilitate the expression and the large-scale purification of various Plasmodium polypeptides.
Animals, Antibodies, Protozoan/immunology, Chromatography, High Pressure Liquid, Dictyostelium/genetics, Dictyostelium/metabolism, Genetic Vectors, Humans, Lectins, Malaria, Vivax/immunology, Malaria, Vivax/parasitology, Merozoite Surface Protein 1/genetics, Merozoite Surface Protein 1/immunology, Plasmodium/genetics, Plasmodium/metabolism, Plasmodium falciparum/genetics, Plasmodium falciparum/metabolism, Plasmodium vivax/genetics, Plasmodium vivax/metabolism, Plasmodium yoelii/genetics, Plasmodium yoelii/immunology, Protozoan Proteins/genetics, Protozoan Proteins/immunology, Recombinant Fusion Proteins/genetics, Recombinant Fusion Proteins/isolation &, purification
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