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https://www.arca.fiocruz.br/handle/icict/1923
IMPROVING THE PRODUCTION OF THE DENATURED RECOMBINANT N-TERMINAL DOMAIN OF RHOPTRY-ASSOCIATED PROTEIN 2 FROM A PLASMODIUM FALCIPARUM TARGET IN THE PATHOLOGY OF ANEMIA IN FALCIPARUM MALARIA
Rhoptry-associated protein 2
6xHis-tagged protein
Purification
Western blotting
Author
Affilliation
Fundação Oswaldo Cruz. Instituto Leônidas e Maria Deane. Manaus, AM, Brasil.
Fundação Oswaldo Cruz. Instituto Leônidas e Maria Deane. Manaus. AM. Brasil.
Universidade de São Paulo. Instituto ciências biomédicas. Departamento de parasitologia. São Paulo. SP, Brasil.
Centro de Pesquisa em Medicina Tropical. Porto Velho, RO, Brasil.
Fundação Oswaldo Cruz. Instituto Leônidas e Maria Deane. Manaus, AM, Brasil.
Fundação Oswaldo Cruz. Instituto Leônidas e Maria Deane. Manaus, AM, Brasil.
Fundação Oswaldo Cruz. Instituto Leônidas e Maria Deane. Manaus. AM. Brasil.
Universidade de São Paulo. Instituto ciências biomédicas. Departamento de parasitologia. São Paulo. SP, Brasil.
Centro de Pesquisa em Medicina Tropical. Porto Velho, RO, Brasil.
Fundação Oswaldo Cruz. Instituto Leônidas e Maria Deane. Manaus, AM, Brasil.
Fundação Oswaldo Cruz. Instituto Leônidas e Maria Deane. Manaus, AM, Brasil.
Abstract
Rhoptry-associated protein 2 (RAP2) is known to be discharged from rhoptry onto the membrane surface of
infected and uninfected erythrocytes (UEs) ex vivo and in vitro and this information provides new insights into the
understanding of the pathology of severe anemia in falciparum malaria. In this study, a hexahistidine-tagged recombinant
protein corresponding to residues 5-190 of the N-terminal of Plasmodium falciparum RAP2 (rN-RAP2)
was produced using a new method of solubilization and purification. Expression was induced with D-lactose, a
less expensive alternative inducer to the more common isopropyl-β-d-thio-galactopyranosidase. The recombinant
protein was purified using two types of commercially-available affinity columns, iminodiacetic and nitrilotriacetic.
rN-RAP2 had immunogenic potential, since it induced high titers of anti-RAP2 antibodies in mice. These antibodies
recognized full-length RAP2 prepared from Triton X-100 extracts from two strains of P. falciparum. In fact, the antibody
recognized a 29-kDa product of RAP2 cleavage as well as 82 and 70-kDa products of RAP1 cleavage. These
results indicate that the two antigens share sequence epitopes. Our expressed protein fragment was shown to contain
a functional epitope that is also present in rhoptry-derived ring surface protein 2 which attaches to the surface of
both infected and UEs and erythroid precursor cells in the bone marrow of malaria patients. Serum from malaria
patients who developed anemia during infection recognized rN-RAP2, suggesting that this protein fragment may be
important for epidemiological studies investigating whether immune responses to RAP2 exacerbate hemolysis in
falciparum malaria patients.
Keywords
Plasmodium falciparumRhoptry-associated protein 2
6xHis-tagged protein
Purification
Western blotting
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