Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: UMLS:C0024530 (malaria)
 44,886 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

A method for purification of a recombinant Plasmodium falciparum protein produced in E. coli and its use in an enzyme-linked immunosorbent assay (ELISA) is described. The cloned gene fragment encodes GLURP,489-1271 the carboxy-terminal 783 amino acid residue portion of a 1271 amino acid residue P. falciparum glutamate rich protein (GLURP), with a molecular weight of 220 kilodalton. The protein is associated with all parasite stages in the human host. Examination of sera from 105 adult Liberians living in a malaria endemic area revealed anti-GLURP IgG antibodies in 98% of the sera. The recombinant GLURP489-1271 was expressed as a chimeric protein, fused with E. coli beta-galactosidase. However, antibodies in sera were directed only against the malaria part of the fusion protein and not against beta-galactosidase. Antigen from in vitro P. falciparum cultures of isolates from Tanzania (F32), Papua New Guinea (MAD20) and Honduras (HB3) completely absorbed specific antibodies, indicating the presence of conserved epitopes produced by all isolates of P. falciparum. Recombinant GLURP489-1271 ELISA is sensitive and rapid, and therefore well-suited for sero-epidemiological studies, and for control of the immunogenicity of a possible future P. falciparum vaccine utilizing epitopes from GLURP.
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PMID:Recombinant Plasmodium falciparum glutamate rich protein; purification and use in enzyme-linked immunosorbent assay. 203 52

The malaria parasite, Plasmodium falciparum, spends part of its life cycle inside the erythrocytes of its human host. In the mature stages of intraerythrocytic growth, the parasite undertakes extensive remodeling of its adopted cellular home by exporting proteins beyond the confines of its own plasma membrane. To examine the signals involved in export of parasite proteins, we have prepared transfected parasites expressing a chimeric protein comprising the N-terminal region of the Plasmodium falciparum exported protein-1 appended to green fluorescent protein. The majority of the population of the chimeric protein appears to be correctly processed and trafficked to the parasitophorous vacuole, indicating that this is the default destination for protein secretion. Some of the protein is redirected to the parasite food vacuole and further degraded. Photobleaching studies reveal that the parasitophorous vacuole contains subcompartments that are only partially interconnected. Dual labeling with the lipid probe, BODIPY-TR-ceramide, reveals the presence of membrane-bound extensions that can bleb from the parasitophorous vacuole to produce double membrane-bound compartments. We also observed regions and extensions of the parasitophorous vacuole, where there is segregation of the lumenal chimera from the lipid components. These regions may represent sites for the sorting of proteins destined for the trafficking to sites beyond the parasitophorous vacuole membrane.
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PMID:The signal sequence of exported protein-1 directs the green fluorescent protein to the parasitophorous vacuole of transfected malaria parasites. 1245 81

Plasmodium falciparum chimeric protein 2 (PfCP-2), fused by erythrocytic stage antigens, AMA-1(III) and MSP1-19, is a potential vaccine candidate against malaria. However, the two-band pattern of this protein product in SDS-PAGE has some negative influence for the application of it for clinical tests. N-terminal sequence analysis of the product showed that the doublet had different N-terminus, with 9 amino acid deletion in the band with low molecular weight. Therefore, the gene was modified to generate a new construct, named PfCP-2.9, which was lack of these 9 residues at its N-terminus. Expression of PfCP-2.9 produced only one band. Moreover, the new construct was as same as the original product in the level of expression, conformation dependence on the disulfide bond, immunogenicity and inhibitory effect on the parasite growth in vitro.
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PMID:[Influence of deleting 9 amino acid residues at N-terminus on immunogenicity of a Plasmodium falciparum chimeric protein]. 1267 88

The C-terminal region (cA) of the major autolysin AcmA of Lactococcus lactis contains three highly similar repeated regions of 45 amino acid residues (LysM domains), which are separated by nonhomologous sequences. The cA domain could be deleted without destroying the cell wall-hydrolyzing activity of the enzyme in vitro. This AcmA derivative was capable neither of binding to lactococcal cells nor of lysing these cells while separation of the producer cells was incomplete. The cA domain and a chimeric protein consisting of cA fused to the C terminus of MSA2, a malaria parasite surface antigen, bound to lactococcal cells specifically via cA. The fusion protein also bound to many other Gram-positive bacteria. By chemical treatment of purified cell walls of L. lactis and Bacillus subtilis, peptidoglycan was identified as the cell wall component interacting with cA. Immunofluorescence studies showed that binding is on specific locations on the surface of L. lactis, Enterococcus faecalis, Streptococcus thermophilus, B. subtilis, Lactobacillus sake, and Lactobacillus casei cells. Based on these studies, we propose that LysM-type repeats bind to peptidoglycan and that binding is hindered by other cell wall constituents, resulting in localized binding of AcmA. Lipoteichoic acid is a candidate hindering component. For L. lactis SK110, it is shown that lipoteichoic acids are not uniformly distributed over the cell surface and are mainly present at sites where no MSA2cA binding is observed.
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PMID:Cell wall attachment of a widely distributed peptidoglycan binding domain is hindered by cell wall constituents. 1268 15

A Plasmodium falciparum chimeric protein 2.9 (PfCP-2.9) was constructed consisting of the C-terminal regions of two leading malaria vaccine candidates, domain III of apical membrane ag-1 (AMA-1) and 19-kDa C-terminal fragment of the merozoite surface protein 1 (MSP1). The PfCP-2.9 was produced by Pichia pastoris in secreted form with a yield of 2600 mg/L and approximately 1 g/L of final product was obtained from a three-step purification process. Analysis of conformational properties of the chimeric protein showed that all six conformational mAbs interacted with the recombinant protein were reduction-sensitive, indicating that fusion of the two cysteine-rich proteins retains critical conformational epitopes. PfCP-2.9 was found to be highly immunogenic in rabbits as well as in rhesus monkeys (Macaca mulatta). The chimeric protein induced both anti-MSP1-19 and anti-AMA-1(III) Abs at levels 11- and 18-fold higher, respectively, than individual components did. Anti-PfCP-2.9 sera from both rabbits and rhesus monkeys almost completely inhibited in vitro growth of the P. falciparum FCC1/HN and 3D7 lines when tested at a 6.7-fold dilution. It was shown that the inhibition is dependent on the presence of Abs to the chimeric protein and their disulfide bond-dependent conformations. Moreover, the activity was mediated by a combination of growth-inhibitory Abs generated by the individual MSP1-19 and AMA-1(III) of PfCP-2.9. The combination of the extremely high yield of the protein and enhancement of its immune response provides a basis to develop an effective and affordable malaria vaccine.
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PMID:Fusion of two malaria vaccine candidate antigens enhances product yield, immunogenicity, and antibody-mediated inhibition of parasite growth in vitro. 1512 4

Following invasion of human erythrocytes, the malaria parasite, Plasmodium falciparum, exports proteins beyond the confines of its own plasma membrane to modify the properties of the host red cell membrane. These modifications are critical to the pathogenesis of malaria. Analysis of the P. falciparum genome sequence has identified a large number of molecules with putative atypical signal sequences. The signals remain poorly characterized; however, a number of molecules with these motifs localize to the host erythrocyte. To examine the role of these atypical signal sequences in the export of parasite proteins, we have generated transfected parasites expressing a chimeric protein comprising the N-terminal region of the P. falciparum ring-infected erythrocyte surface antigen (RESA) appended to green fluorescent protein (GFP). This N-terminal region contains a hydrophobic stretch of amino acids that is presumed to act as a noncanonical secretory signal sequence. Modulation of the timing of transgene expression demonstrates that trafficking of malaria proteins into the host erythrocyte is dependent on both the presence of an appropriate transport signal and the timing of expression. Transgene expression under the control of a trophozoite-specific promoter mistargets the chimeric molecule to the parasitophorous vacuole surrounding the parasite. However, expression of RESA-GFP in schizont stages, under the control of the RESA promoter, enables correct trafficking of a population of the chimeric protein to the host erythrocyte.
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PMID:Correct promoter control is needed for trafficking of the ring-infected erythrocyte surface antigen to the host cytosol in transfected malaria parasites. 1538 14

Because invasion of erythrocytes by Plasmodium falciparum merozoites involves multiple receptor-ligand interactions, it may be necessary to develop a multivalent malaria vaccine that is comprised of distinct parasite ligands. PfAMA-1, PfMSP1, and PfEBA-175 are merozoite proteins that play important roles in invasion. We have constructed a PfCP-2.9 chimeric protein consisting of PfAMA-1 and PfMSP1 and tested it for immunogenicity in animal models and humans. The F2 subdomain of PfEBA-175 (PfEBA-175II F2) was identified as the binding domain for glycophorin A on erythrocytes. In this study, we used the codon frequencies of the yeast Pichia pastoris to redesign and synthesize a gene encoding the F2 domain. We found that the codon-optimized gene was expressed at a high level in P. pastoris as a soluble protein with a yield of about 300 mg/liter. The expressed protein was able to bind normal erythrocytes but not those treated with neuraminidase or trypsin. Moreover, the protein was recognized by the sera of malaria patients and was highly immunogenic in mice, rabbits, and rhesus monkeys. Immunoglobulin G isolated from both immunized rabbits and monkeys inhibited in vitro parasite growth. Immunization of animals with a combination of PfEBA-175II F2 and PfCP-2.9 did not result in antigen (Ag) competition in animals. Moreover, antibodies to both PfEBA-175II F2 and PfCP-2.9, isolated from rabbits immunized with both constructs, inhibited parasite growth in vitro. The combination of high yield, functional folding, antibody inhibition, and lack of Ag competition provides support for inclusion of these merozoite proteins in a combination vaccine against infection with blood-stage parasites.
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PMID:Evaluation of three Pichia pastoris-expressed Plasmodium falciparum merozoite proteins as a combination vaccine against infection with blood-stage parasites. 1617 27

Antibodies against the malaria vaccine candidate apical membrane antigen-1 (AMA-1) can inhibit invasion of merozoites into RBC, but antigenic diversity can compromise vaccine efficacy. We hypothesize that polymorphic sites located within inhibitory epitopes function as antigenic escape residues (AER). By using an in vitro model of antigenic escape, the inhibitory contribution of 24 polymorphic sites of the 3D7 AMA-1 vaccine was determined. An AER cluster of 13 polymorphisms, located within domain 1, had the highest inhibitory contribution. Within this AER cluster, antibodies primarily targeted five polymorphic residues situated on an alpha-helical loop. A second important AER cluster was localized to domain 2. Domain 3 polymorphisms enhanced the inhibitory contribution of the domain 2 AER cluster. Importantly, the AER clusters could be split, such that chimeras containing domain 1 of FVO and domain 2 + 3 of 3D7 generated antisera that showed similarly high level inhibition of the two vaccine strains. Antibodies to this chimeric protein also inhibited unrelated strains of the parasite. Interstrain AER chimeras can be a way to incorporate inhibitory epitopes of two AMA-1 strains into a single protein. The AER clusters map in close proximity to conserved structural elements: the hydrophobic trough and the C-terminal proteolytic processing site. This finding led us to hypothesize that a conserved structural basis of antigenic escape from anti-AMA-1 exists. Genotyping high-impact AER may be useful for classifying AMA-1 strains into inhibition groups and to detect allelic effects of an AMA-1 vaccine in the field.
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PMID:Structural basis of antigenic escape of a malaria vaccine candidate. 1763 23

Immune response against circumsporozoite protein (CSP) of Plasmodium berghei, a major surface protein on the sporozoite, confers protection in various murine malaria models. Engineered DNA vaccine encoding CSP and 3 copies of C3d caused an unexpected loss in protection attributed to the binding of C3d to the C-terminal region of CSP. Because the C3d region known as p28 represents the complement receptor (CR) 2-binding motif, we developed a CSP-3 copies of p28 DNA construct (CSP-3p28). CSP-3p28-immunized mice were better protected against P. berghei sporozoites than CSP-immunized mice 6 weeks after the 2nd boost, produced sufficient IgG1 anti-CSP and CSP C-terminus antibody and failed to produce IgG2a. CSP-3C3d-immunized mice were not protected, failed to produce IgG1 and produced high amounts of IgG2a. We conclude that use of the CR2-binding motif of C3d as molecular adjuvant to CSP results in anti-malaria protective immune response probably by targeting the chimeric protein to CR2.
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PMID:C3d-defined complement receptor-binding peptide p28 conjugated to circumsporozoite protein provides protection against Plasmodium berghei. 1793 54

Developing a polyepitope vaccine, a chimeric protein which contains diverse types of antigenic epitopes, is a promising strategy to prevent malaria. Previously, we had constructed a library of polyeptitope chimeric genes against Plasmodium falciparum without any protein tags. In an attempt to develop an efficient and universal procedure for purification of polyepitope chimeric proteins, we assembled an immunoaffinity chromatography (IAC) column with affinity-purified specific polyclonal IgY (mpIgY) antibodies that recognized the same C-terminal epitope tag of chimeric proteins in the library. A single-step and universal protocol was established and successfully applied for the purification of chimeric proteins. Using this protocol, chimeric proteins were specifically purified from an Escherichia coli expression system, and the purity and authenticity were verified by gel electrophoresis and Western blot analysis. Moreover, the comparison between this IAC method and the conventional chromatography, using two anion exchange columns followed by a step of gel filtration, showed that the new method was more efficient with an 8-fold greater yield. The results suggest that this IAC method will be an efficient approach for the purifications of polyepitope vaccine candidates against P. falciparum in our future study, and also be valuable for other similar applications.
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PMID:Immunoaffinity purification of polyepitope proteins against Plasmodium falciparum with chicken IgY specific to their C-terminal epitope tag. 2094 54


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