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

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 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.Key words: Plasmodium falciparum -rhoptry-associated protein 2 -6xHis-tagged protein -purification -Western blotting Rhoptry-associated protein 2 (RAP2) is a 398-amino acid protein encoded by a single exon located on chromosome 5 of Plasmodium falciparum (Perez-Leal et al. 2005). It is discharged from rhoptry, an apical organelle of the merozoite, onto the surface of the erythrocyte membrane during the blood stage of malaria infections. This discharge leads to the formation of a parasitophorous vacuole (PV) membrane. RAP2 is considered to be an important candidate for vaccine development, since monkeys immunized with RAP2 were partially protected against parasite challenge (Perrin et al. 1985, Ridley et al. 1990 response plays a role in the pathology of severe anemia by inducing lysis of UEs immediately after discharge of this antigen onto the surface membrane. Saul et al. (1992) cloned the entire RAP2 gene and described its expression and purification using a hexahistidine system. Their methods yielded a low level of expression; further, the purification of RAP2 presented a range of difficulties due to the protein's lipophilicity. Expression in their study was induced using isopropyl-β-d-thio-galactopyranosidase (IPTG), which substantially raised the cost of protein production due to the many liters of culture necessary to obtain a reasonable amount of protein. The authors described that RAP2 precipitates even at very low concentrations in the absence of a strong detergent; 6 M guanidine was needed to solubilize inclusion bodies. Furthermore, Saul et al. (1992) reported that recombinant RAP2 could be eluted effectively only with a denaturation buffer containing 0.5 M imidazole (iDB) and not with a pH gradient.In the present study, a hexahistidine-tagged RAP2 fragment containing the N-terminal (N-RAP2) residues 5-190 of P. falciparum RAP2 was produced. We achieved efficient induction of expression in Escheria coli using d-lactose as a less expensive alternative to IPTG. Furthermore, we compared the purifying power of two types of commercially-available affinity columns, iminodiacetic (Ni-IDA) and nitrilotriacetic (Ni-

Antibodies to the Plasmodium falciparum rhoptry protein RAP‐2/RSP‐2 in relation to anaemia in Cameroonian children

“…As a result of the hydrophobic nature of the RAP‐2/RSP‐2 antigen (35,37), it has, until recently, not been possible to produce a recombinant protein that could be used in ELISA for the determinations of antibodies (35). Thus, initially, in our study for the determination of the levels of RAP‐2/RSP‐2‐reactive antibodies, the binding of antibodies to RAP‐2/RSP‐2‐tagged RBCs was analysed by flow cytometry, using serum samples pre‐adsorbed on infected RBCs lacking the expression of RAP‐2/RSP‐2 on their surface (25).…”

“…Naturally occurring antibody responses to P. falciparum RAP‐2 were measured against a recombinant RAP‐2 (35) by ELISA and against the whole parasite antigen using parasites from infected cultures (here considered to as native RAP‐2) by flow cytometry.…”

Novel Cinchona Alkaloid Derivatives as Potential Antimalarial Agents through Receptor–Inhibitor Interaction Fingerprint and Biosynthesis Design

“…Therefore, several attempts have been started to identify clinical targets for healing human malarial disease. Molecular modeling [19][20][21][22] and database formation approaches as new techniques are currently in the innovation development and execution stages [23][24][25][26] to enhance potential antimalarial targets [27][28][29][30][31] .…”