- Open Access
Towards a comprehensive Plasmodium falciparum merozoite cell surface and secreted recombinant protein library
© Zenonos et al.; licensee BioMed Central Ltd. 2014
- Received: 12 December 2013
- Accepted: 7 March 2014
- Published: 12 March 2014
Plasmodium falciparum is the aetiological agent for malaria, a deadly infectious disease for which no vaccine has yet been licensed. The proteins displayed on the merozoite cell surface have long been considered attractive vaccine targets because of their direct exposure to host antibodies; however, progress in understanding the functional role of these targets has been hindered by technical challenges associated with expressing these proteins in a functionally active recombinant form. To address this, a method that enables the systematic expression of functional extracellular Plasmodium proteins was previously developed, and used to create a library of 42 merozoite proteins.
To compile a more comprehensive library of recombinant proteins representing the repertoire of P. falciparum merozoite extracellular proteins for systematic vaccine and functional studies, genome-wide expression profiling was used to identify additional candidates. Candidate proteins were recombinantly produced and their integrity and expression levels were tested by Western blotting and ELISA.
Twenty-five additional genes that were upregulated during late schizogony, and predicted to encode secreted and cell surface proteins, were identified and expressed as soluble recombinant proteins. A band consistent with the entire ectodomain was observed by immunoblotting for the majority of the proteins and their expression levels were quantified. By using sera from malaria-exposed immune adults, the immunoreactivity of 20 recombinant proteins was assessed, and most of the merozoite ligands were found to carry heat-labile epitopes. To facilitate systematic comparative studies across the entire library, multiple Plasmodium proteins were simultaneously purified using a custom-made platform.
A library of recombinant P. falciparum secreted and cell surface proteins was expanded by 20 additional proteins, which were shown to express at usable levels and contain conformational epitopes. This resource of extracellular P. falciparum merozoite proteins, which now contains 62 full-length ectodomains, will be a valuable tool in elucidating the function of these proteins during the blood stages of infection, and facilitate the comparative assessment of blood stage vaccine candidates.
- Plasmodium falciparum
- Recombinant proteins
- Mammalian expression system
- Protein purification platform
Plasmodium falciparum is the aetiological agent of the most deadly form of malaria, an infectious tropical disease that accounts for up to one million deaths annually[1, 2]. The vast majority of malaria fatalities (85-90%) occur in sub-Saharan Africa, primarily in pregnant women and children under the age of five[2, 3]. While anti-malarial drugs exist, the emergence of drug-resistant parasite strains remains a global health concern and no vaccine has been licensed to date.
The asexual blood stages of malaria are initiated when a form of the parasite, called a merozoite, invades, replicates and synchronously ruptures host erythrocytes releasing up to 32 progeny merozoites that can invade new erythrocytes. This cyclical phase causes the recurrent fevers and chills that are characteristic of malaria infection. Merozoites are ovoid cells containing apically located secretory organelles that release proteins which are required for the invasion of new erythrocytes[6, 7]. While erythrocyte invasion is a rapid process, the brief extracellular exposure of merozoites outside of their intra-erythrocytic niche places them in direct contact with host antibodies, which contribute to naturally acquired immunity to malaria[8, 9]; therefore, merozoite cell surface and secreted proteins have long been considered attractive targets for rational vaccine development.
The publication of the P. falciparum genome project in 2002 identified the full complement of parasite proteins but progress in understanding the function of these proteins, including those displayed on the merozoite cell surface, has been hindered by the technical difficulties in expressing Plasmodium proteins in a functionally active form. Although the reasons why Plasmodium proteins are difficult to express in heterologous expression systems are not clear, several protein characteristics, such as high molecular mass (>60 kDa), presence of export motifs, and atypical signal peptide sequences negatively impact recombinant expression. In addition, the remarkably high (~80%) A + T content of parasite genes can result in long stretches of repetitive amino acids, and codons that are not frequently used by organisms popular for heterologous protein expression. Extracellular vaccine candidates, in particular, present an additional challenge because they often require structurally critical disulfide bonds for correct folding and contain transmembrane domains that make them difficult to solubilize in detergents that retain their native conformation[14–16].
Despite these challenges, recombinant expression of Plasmodium proteins has been attempted in a number of expression systems[12, 17] ranging from bacteria, yeast[14, 19], Dictyostelium discoideum, plants and algae[21, 22] to mammalian cells[23, 24] and cell-free systems. Among them, Escherichia coli is the most popular, but the systematic expression of functional P. falciparum proteins remains difficult, with success rates as low as just 6%, and often requires subsequent laborious and complex refolding procedures with uncertain outcomes. Consequently, the functional characterization of extracellular parasite proteins has typically been restricted to smaller subfragments that can be expressed rather than the full-length protein or entire ectodomain, which is more likely to be representative of the native protein.
The development of a standardized method to express large panels of P. falciparum cell surface and secreted proteins in their native conformation would enable comprehensive protein libraries to be systematically screened in parallel so that direct comparisons between antigens can be made in functional assays such as vaccine screening and immuno-epidemiology studies. To achieve this, Crosnier and colleagues recently developed a method of expressing the entire ectodomains of functional recombinant Plasmodium proteins and used it to compile a large library of 42 proteins. Working towards a comprehensive library of cell surface and secreted P. falciparum merozoite proteins, this manuscript describes the identification and characterization of an additional 20 proteins.
Recombinant protein design and expression
Proteins were expressed essentially as described previously. Briefly, full-length secreted molecules and the entire ectodomains of membrane-embedded proteins were identified using transmembrane, GPI-anchor, and signal peptide prediction software. To prevent the inappropriate addition of glycans, which are absent from Plasmodium proteins, all potential N-linked glycosylation sites (N-X-S/T, where X is not proline) were systematically mutated by substituting alanine for serine/threonine at these sites. Gene constructs were made by gene synthesis (GeneartAG) using sequences that were codon-optimized for expression in human cells. Protein coding sequences were flanked with unique NotI and AscI restriction sites and subcloned into a derivative of the pTT3 expression plasmid between a 5’ mouse variable κ light chain signal peptide, and a 3’ tag consisting of the rat Cd4 domains 3 and 4 followed by an enzymatic biotinylation sequence and a hexahistidine tag. Proteins were expressed as soluble monobiotinylated proteins by transient cotransfection of HEK293E cells with the BirA biotin ligase and harvested six days post transfection. All expression plasmids are openly available from Addgene.
Parallel protein purification
His-tagged recombinant merozoite proteins were purified from spent tissue culture supernatants by using a custom-built, piston-driven, sample-loading apparatus as described previously. Briefly, a 96-well His MultiTrap HP filter plate (GE Healthcare) was pre-equilibrated with binding buffer (20 mM sodium phosphate, 40 mM imidazole, 0.5 M NaCl, pH 7.4) at a flow rate of 1 mL/min. The harvested tissue culture supernatants (~200 mL for each protein) were supplemented with imidazole (10 mM) before loading each well at 1 mL/min. The plate was washed with 1.6 mL of binding buffer and proteins eluted with 0.2 mL of elution buffer (20 mM sodium phosphate, 0.4 M imidazole, 0.5 M NaCl, pH 7.4).
Enzyme-linked immunosorbent assay (ELISA)
ELISAs were performed as previously described. Briefly, purified recombinant biotinylated proteins were serially diluted in PBS-T (PBS, 0.1% Tween-20) with 2% BSA, and captured on streptavidin-coated, 96-well plates (NUNC) for one hour before washing and incubating with the anti-Cd4 monoclonal antibody OX68 (1 μg/mL in PBS-T, 2% BSA) for another hour. Plates were washed and incubated with an anti-mouse IgG (Sigma) secondary antibody conjugated to alkaline phosphatase for one hour before further washes and incubation with p-nitrophenyl phosphate (Substrate 104; Sigma) at 1 mg/mL. Absorbance was measured at 405 nm on a PHERAstar plus (BMG Labtech). Concentrations were calculated by comparison to known standards.
Each purified recombinant protein was resolved by SDS-PAGE under reducing conditions before blotting onto Hybond-P PVDF membrane (GE Healthcare) for one hour at 30 V. Membranes were blocked with 2% BSA, in PBS-T and incubated with 0.02 μg/mL of streptavidin-HRP (Jackson Immunoresearch) diluted in PBS-T, 0.2% BSA and detected with the Supersignal West pico chemiluminescent substrate (Pierce).
Proteins were heat treated at 80°C for 10 minutes or left untreated and immobilized on streptavidin-coated, 96-well plates (NUNC), at concentrations sufficient for complete saturation of the available binding surface/well (as determined by ELISA). Following three washes in PBS-T, plates were incubated with pooled sera from malaria-exposed Malawian adults or malaria-naïve UK individuals at a 1:1,000 dilution in PBS-T, 2% BSA followed by an alkaline phosphatase-conjugated anti-human IgG secondary antibody (Sigma) and detected as above.
Identification of candidate Plasmodium falciparum merozoite cell surface and secreted proteins
The putative merozoite cell surface and secreted proteins that were chosen for recombinant expression
Region targeted for recombinant expression
rhoptry-associated membrane antigen (RAMA)
Conserved Plasmodium protein, unknown function
Conserved Plasmodium protein, unknown function
Conserved Plasmodium protein, unknown function
Merozoite-associated tryptophan-rich antigen, putative
LCCL domain-containing protein
Conserved Plasmodium protein, unknown function
Conserved Plasmodium protein, unknown function
Cysteine-rich protective antigen (CyRPA)
To design the expression constructs, the entire predicted extracellular domain was selected between the signal sequence and the GPI-anchor, if present. Any predicted N-linked glycosylation sites were systematically removed by substituting alanine for serine/threonine at these sites. Expression constructs were made by gene synthesis and codon optimized for expression in human cells. All expression plasmids are publicly available through Addgene, a not-for-profit, open access plasmid repository.
Expression and purification of an expanded Plasmodium falciparum merozoite protein library
All proteins were expressed in HEK293E cells as soluble fusion proteins that contained a C-terminal rat Cd4(d3 + 4)-hexahistidine tag for purification and could be optionally monobiotinylated by cotransfecting a secreted version of the E. coli BirA enzyme. To purify many proteins in parallel for comparative screening, a custom-built protein purification system was employed, that enables the simultaneous purification of up to 96 His-tagged proteins, even from large (>50 mL) tissue culture volumes.
Following purification, recombinant proteins were quantitated by ELISA and while expression levels varied significantly between individual proteins, most were purified to micromolar levels (Table 1). Overall, detectable expression was obtained for 22 out of 25 (88%) proteins. Both GLURP and SERA6, although detected by ELISA, were expressed at levels that were too low to include in further analysis and three proteins (PF14_0044, PFA0445w, SERA1) remained undetectable by ELISA despite repeated transfections.
Members of the recombinant merozoite protein library proteins are immunoreactive and carry heat-labile epitopes
To demonstrate that serum antibodies were recognizing conformational epitopes within the protein library, all recombinant proteins were denatured by heat treatment before being captured via their biotin tag. For 16 of the 20 proteins (all but MSRP4, SERA7, PFA0210c, and PFB0475c) the immunoreactivity decreased significantly when the proteins were heat inactivated, establishing that the antigens contain heat-labile epitopes. These data show that the merozoite recombinant proteins are correctly folded and at least in part mimic the native protein conformation.
The technical difficulties in expressing Plasmodium proteins in a recombinant functional form has presented difficulties both for basic malaria research and vaccine development. Here, an approach based on a mammalian expression system has been utilized to significantly expand a recombinant library consisting of recombinant P. falciparum merozoite extracellular proteins from 42 to 62 members. A high-throughput, custom-made purification platform was successfully used to purify recombinant proteins from large volumes of tissue culture supernatants to permit systematic comparative studies of purified antigens.
While the overall success rate of expression was high, consistent with previous experience of the HEK293 system[27, 53–57], a few merozoite proteins still failed to express, or were expressed at low levels. Although the genes were codon-optimized for human cells, P. falciparum proteins are unusual because they are enriched in asparagine, glutamic acid and lysine, and often contain homopolymeric stretches of amino acids. Indeed, it has been recently demonstrated that the stability of several Plasmodium proteins depends upon their association with heat shock proteins which act as molecular chaperones. Therefore, recombinant protein expression in the HEK293 system could be further enhanced by the presence of Pf Hsp110c, which has been proposed to be a protein-stabilizing chaperone.
Using sera from malaria-immune adults, it has been shown that the expressed recombinant proteins contained heat-labile epitopes suggesting that they adopt their native conformation and are likely to be biochemically active. A number of proteins (e g, PF10_0166, PFA0135w) showed little serological response in comparison to the negative control. While these proteins may not contain epitopes present in the native protein, the possibility that the native proteins are poorly immunogenic and do not induce a strong antibody response in vivo, cannot be excluded. For example, previous studies reported that only 23% of immune sera examined contained specific serum IgG antibodies against PFA0135w, suggesting that this protein does not normally elicit strong humoral responses. Interestingly, RIPR, which binds to Pf RH5, is also among the group of generally low responders. Pf RH5 is a high priority vaccine target as it plays an essential and universal role in erythrocyte invasion[53, 57, 61–63], yet it is poorly immunogenic in vivo, possibly due to its late release onto the merozoite surface during erythrocyte invasion. RIPR may be similarly masked from the host immune system, but further work with this antigen is clearly required.
A library of recombinant P. falciparum proteins has been expanded and characterized with the eventual aim of compiling a set that is representative of the merozoite surface. These plasmids, which are freely available to the global research community through Addgene, will be a valuable resource for basic research and aid the efforts to develop an effective malaria vaccine.
This work was supported by the Wellcome Trust grant number . We thank Cécile Crosnier for helpful comments on the manuscript and Faith Osier and Kevin Marsh for malaria immune sera.
- Murray CJL, Rosenfeld LC, Lim SS, Andrews KG, Foreman KJ, Haring D, Fullman N, Naghavi M, Lozano R, Lopez AD: Global malaria mortality between 1980 and 2010: a systematic analysis. Lancet. 2012, 379: 413-431. 10.1016/S0140-6736(12)60034-8.View ArticlePubMedGoogle Scholar
- World Health Organization: World Malaria Report: 2012. 2012, Geneva: Switzerland: Global Malaria ProgrammeGoogle Scholar
- Geels MJ, Imoukhuede EB, Imbault N, van Schooten H, McWade T, Troye-Blomberg M, Dobbelaer R, Craig AG, Leroy O: European vaccine initiative: lessons from developing malaria vaccines. Expert Rev Vaccines. 2011, 10: 1697-1708. 10.1586/erv.11.158.View ArticlePubMedGoogle Scholar
- Miller LH, Baruch DI, Marsh K, Doumbo OK: The pathogenic basis of malaria. Nature. 2002, 415: 673-679. 10.1038/415673a.View ArticlePubMedGoogle Scholar
- Chen Q, Schlichtherle M, Wahlgren M: Molecular aspects of severe malaria. Clin Microbiol Rev. 2000, 13: 439-450. 10.1128/CMR.13.3.439-450.2000.PubMed CentralView ArticlePubMedGoogle Scholar
- Garcia CRS, de Azevedo MF, Wunderlich G, Budu A, Young JA, Bannister L: Plasmodium in the postgenomic era: new insights into the molecular cell biology of malaria parasites. Int Rev Cell Mol Biol. 2008, 266: 85-156.View ArticlePubMedGoogle Scholar
- Dvorak JA, Miller LH, Whitehouse WC, Shiroishi T: Invasion of erythrocytes by malaria merozoites. Science. 1975, 187: 748-750. 10.1126/science.803712.View ArticlePubMedGoogle Scholar
- Cohen S, McGregor IA, Carrington S: Gamma-globulin and acquired immunity to human malaria. Nature. 1961, 192: 733-737. 10.1038/192733a0.View ArticlePubMedGoogle Scholar
- Cohen S, Butcher GA, Crandall RB: Action of malarial antibody in vitro. Nature. 1969, 223: 368-371. 10.1038/223368a0.View ArticlePubMedGoogle Scholar
- Gardner MJ, Hall N, Fung E, White O, Berriman M, Hyman RW, Carlton JM, Pain A, Nelson KE, Bowman S, Paulsen IT, James K, Eisen JA, Rutherford K, Salzberg SL, Craig A, Kyes S, Chan M-S, Nene V, Shallom SJ, Suh B, Peterson J, Angiuoli S, Pertea M, Allen J, Selengut J, Haft D, Mather MW, Vaidya AB, Martin DMA: Genome sequence of the human malaria parasite Plasmodium falciparum. Nature. 2002, 419: 498-511. 10.1038/nature01097.View ArticlePubMedGoogle Scholar
- Bartholdson SJ, Crosnier C, Bustamante LY, Rayner JC, Wright GJ: Identifying novel Plasmodium falciparum erythrocyte invasion receptors using systematic extracellular protein interaction screens. Cell Microbiol. 2013, 15: 1304-1312. 10.1111/cmi.12151.PubMed CentralView ArticlePubMedGoogle Scholar
- Birkholtz L-M, Blatch G, Coetzer TL, Hoppe HC, Human E, Morris EJ, Ngcete Z, Oldfield L, Roth R, Shonhai A, Stephens L, Louw AI: Heterologous expression of plasmodial proteins for structural studies and functional annotation. Malar J. 2008, 7: 197-10.1186/1475-2875-7-197.PubMed CentralView ArticlePubMedGoogle Scholar
- Tsuboi T, Takeo S, Iriko H, Jin L, Tsuchimochi M, Matsuda S, Han E-T, Otsuki H, Kaneko O, Sattabongkot J, Udomsangpetch R, Sawasaki T, Torii M, Endo Y: Wheat germ cell-free system-based production of malaria proteins for discovery of novel vaccine candidates. Infect Immun. 2008, 76: 1702-1708. 10.1128/IAI.01539-07.PubMed CentralView ArticlePubMedGoogle Scholar
- Stowers AW, Zhang Y, Shimp RL, Kaslow DC: Structural conformers produced during malaria vaccine production in yeast. Yeast. 2001, 18: 137-150. 10.1002/1097-0061(20010130)18:2<137::AID-YEA657>3.0.CO;2-X.View ArticlePubMedGoogle Scholar
- Vedadi M, Lew J, Artz J, Amani M, Zhao Y, Dong A, Wasney G, Gao M, Hills T, Brokx S, Qiu W, Sharma S, Diassiti A, Alam Z, Melone M, Mulichak A, Wernimont A, Bray J, Loppnau P, Plotnikova O, Newberry K, Sundararajan E, Houston S, Walker J, Tempel W, Bochkarev A, Kozieradzki I, Edwards A, Arrowsmith C, Roos D: Genome-scale protein expression and structural biology of Plasmodium falciparum and related Apicomplexan organisms. Mol Biochem Parasitol. 2007, 151: 100-110. 10.1016/j.molbiopara.2006.10.011.View ArticlePubMedGoogle Scholar
- Wright GJ: Signal initiation in biological systems: the properties and detection of transient extracellular protein interactions. Mol Biosyst. 2009, 5: 1405-1412. 10.1039/b903580j.PubMed CentralView ArticlePubMedGoogle Scholar
- Fernández-Robledo JA, Vasta GR: Production of recombinant proteins from protozoan parasites. Trends Parasitol. 2010, 26: 244-254. 10.1016/j.pt.2010.02.004.PubMed CentralView ArticlePubMedGoogle Scholar
- Outchkourov NS, Roeffen W, Kaan A, Jansen J, Luty A, Schuiffel D, van Gemert GJ, van de Vegte-Bolmer M, Sauerwein RW, Stunnenberg HG: Correctly folded Pf s48/45 protein of Plasmodium falciparum elicits malaria transmission-blocking immunity in mice. Proc Natl Acad Sci U S A. 2008, 105: 4301-4305. 10.1073/pnas.0800459105.PubMed CentralView ArticlePubMedGoogle Scholar
- Tsai CW, Duggan PF, Shimp RL, Miller LH, Narum DL: Overproduction of Pichia pastoris or Plasmodium falciparum protein disulfide isomerase affects expression, folding and O-linked glycosylation of a malaria vaccine candidate expressed in P. pastoris. J Biotechnol. 2006, 121: 458-470. 10.1016/j.jbiotec.2005.08.025.View ArticlePubMedGoogle Scholar
- Van Bemmelen MX, Beghdadi-Rais C, Desponds C, Vargas E, Herrera S, Reymond CD, Fasel N: Expression and one-step purification of Plasmodium proteins in Dictyostelium. Mol Biochem Parasitol. 2000, 111: 377-390. 10.1016/S0166-6851(00)00330-3.View ArticlePubMedGoogle Scholar
- Ghosh S, Malhotra P, Lalitha P: Expression of Plasmodium falciparum C-terminal region of merozoite surface protein (Pf MSP1 19), a potential malaria vaccine candidate, in. Plant Sci. 2002, 162: 335-343. 10.1016/S0168-9452(01)00555-6.View ArticleGoogle Scholar
- Gregory J a, Li F, Tomosada LM, Cox CJ, Topol AB, Vinetz JM, Mayfield S: Algae-produced Pf s25 elicits antibodies that inhibit malaria transmission. PLoS One. 2012, 7: e37179-10.1371/journal.pone.0037179.PubMed CentralView ArticlePubMedGoogle Scholar
- VanBuskirk KM, Sevova E, Adams JH: Conserved residues in the Plasmodium vivax Duffy-binding protein ligand domain are critical for erythrocyte receptor recognition. Proc Natl Acad Sci U S A. 2004, 101: 15754-15759. 10.1073/pnas.0405421101.PubMed CentralView ArticlePubMedGoogle Scholar
- Tolia NH, Enemark EJ, Sim BKL, Joshua-Tor L: Structural basis for the EBA-175 erythrocyte invasion pathway of the malaria parasite Plasmodium falciparum. Cell. 2005, 122: 183-193. 10.1016/j.cell.2005.05.033.View ArticlePubMedGoogle Scholar
- Mehlin C, Boni E, Buckner FS, Engel L, Feist T, Gelb MH, Haji L, Kim D, Liu C, Mueller N, Myler PJ, Reddy JT, Sampson JN, Subramanian E, Van Voorhis WC, Worthey E, Zucker F, Hol WGJ: Heterologous expression of proteins from Plasmodium falciparum: results from 1000 genes. Mol Biochem Parasitol. 2006, 148: 144-160. 10.1016/j.molbiopara.2006.03.011.View ArticlePubMedGoogle Scholar
- De Marco A: Strategies for successful recombinant expression of disulfide bond-dependent proteins in Escherichia coli. Microb Cell Fact. 2009, 8: 26-10.1186/1475-2859-8-26.PubMed CentralView ArticlePubMedGoogle Scholar
- Crosnier C, Wanaguru M, McDade B, Osier FH, Marsh K, Rayner JC, Wright GJ: A library of functional recombinant cell-surface and secreted P. falciparum merozoite proteins. Mol Cell Proteomics. 2013, 12: 3976-3986. 10.1074/mcp.O113.028357.PubMed CentralView ArticlePubMedGoogle Scholar
- Sonnhammer EL, von Heijne G, Krogh A: A hidden Markov model for predicting transmembrane helices in protein sequences. Proc Int Conf Intell Syst Mol Biol. 1998, 6: 175-182.PubMedGoogle Scholar
- Eisenhaber B, Bork P, Eisenhaber F: Prediction of potential GPI-modification sites in proprotein sequences. J Mol Biol. 1999, 292: 741-758. 10.1006/jmbi.1999.3069.View ArticlePubMedGoogle Scholar
- Petersen TN, Brunak S, von Heijne G, Nielsen H: SignalP 4.0: discriminating signal peptides from transmembrane regions. Nat Methods. 2011, 8: 785-786. 10.1038/nmeth.1701.View ArticlePubMedGoogle Scholar
- Dieckmann-Schuppert A, Bender S, Odenthal-Schnittler M, Bause E, Schwarz RT: Apparent lack of N-glycosylation in the asexual intraerythrocytic stage of Plasmodium falciparum. Eur J Biochem. 1992, 205: 815-825. 10.1111/j.1432-1033.1992.tb16846.x.View ArticlePubMedGoogle Scholar
- Crosnier C, Staudt N, Wright GJ: A rapid and scalable method for selecting recombinant mouse monoclonal antibodies. BMC Biol. 2010, 8: 76-10.1186/1741-7007-8-76.PubMed CentralView ArticlePubMedGoogle Scholar
- Sun Y, Gallagher-Jones M, Barker C, Wright GJ: A benchmarked protein microarray-based platform for the identification of novel low-affinity extracellular protein interactions. Anal Biochem. 2012, 424: 45-53. 10.1016/j.ab.2012.01.034.PubMed CentralView ArticlePubMedGoogle Scholar
- Durocher Y, Perret S, Kamen A: High-level and high-throughput recombinant protein production by transient transfection of suspension-growing human 293-EBNA1 cells. Nucleic Acids Res. 2002, 30: E9-10.1093/nar/30.2.e9.PubMed CentralView ArticlePubMedGoogle Scholar
- Kerr JS, Wright GJ: Avidity-based extracellular interaction screening (AVEXIS) for the scalable detection of low-affinity extracellular receptor-ligand interactions. J Vis Exp. 2012, 7: e3881-Google Scholar
- Herscovitch M, Perkins E, Baltus A, Fan M: Addgene provides an open forum for plasmid sharing. Nat Biotechnol. 2012, 30: 316-317. 10.1038/nbt.2177.View ArticlePubMedGoogle Scholar
- Bushell KM, Söllner C, Schuster-Boeckler B, Bateman A, Wright GJ: Large-scale screening for novel low-affinity extracellular protein interactions. Genome Res. 2008, 18: 622-630. 10.1101/gr.7187808.PubMed CentralView ArticlePubMedGoogle Scholar
- Llinás M, Bozdech Z, Wong ED, Adai AT, DeRisi JL: Comparative whole genome transcriptome analysis of three Plasmodium falciparum strains. Nucleic Acids Res. 2006, 34: 1166-1173. 10.1093/nar/gkj517.PubMed CentralView ArticlePubMedGoogle Scholar
- Bozdech Z, Llinás M, Pulliam BL, Wong ED, Zhu J, DeRisi JL: The transcriptome of the intraerythrocytic developmental cycle of Plasmodium falciparum. PLoS Biol. 2003, 1: E5-PubMed CentralView ArticlePubMedGoogle Scholar
- Tham W-H, Healer J, Cowman AF: Erythrocyte and reticulocyte binding-like proteins of Plasmodium falciparum. Trends Parasitol. 2012, 28: 23-30. 10.1016/j.pt.2011.10.002.View ArticlePubMedGoogle Scholar
- Cowman AF, Crabb BS: Invasion of red blood cells by malaria parasites. Cell. 2006, 124: 755-766. 10.1016/j.cell.2006.02.006.View ArticlePubMedGoogle Scholar
- Aurrecoechea C, Brestelli J, Brunk BP, Dommer J, Fischer S, Gajria B, Gao X, Gingle A, Grant G, Harb OS, Heiges M, Innamorato F, Iodice J, Kissinger JC, Kraemer E, Li W, Miller JA, Nayak V, Pennington C, Pinney DF, Roos DS, Ross C, Stoeckert CJ, Treatman C, Wang H: PlasmoDB: a functional genomic database for malaria parasites. Nucleic Acids Res. 2009, 37 (Database issue): D539-D543.PubMed CentralView ArticlePubMedGoogle Scholar
- Punta M, Coggill PC, Eberhardt RY, Mistry J, Tate J, Boursnell C, Pang N, Forslund K, Ceric G, Clements J, Heger A, Holm L, Sonnhammer ELL, Eddy SR, Bateman A, Finn RD: The Pfam protein families database. Nucleic Acids Res. 2012, 40 (Database issue): D290-D301.PubMed CentralView ArticlePubMedGoogle Scholar
- Singh S, Soe S, Weisman S, Barnwell JW, Pérignon JL, Druilhe P: A conserved multi-gene family induces cross-reactive antibodies effective in defense against Plasmodium falciparum. PLoS One. 2009, 4: e5410-10.1371/journal.pone.0005410.PubMed CentralView ArticlePubMedGoogle Scholar
- Burgess BR, Schuck P, Garboczi DN: Dissection of merozoite surface protein 3, a representative of a family of Plasmodium falciparum surface proteins, reveals an oligomeric and highly elongated molecule. J Biol Chem. 2005, 280: 37236-37245. 10.1074/jbc.M506753200.View ArticlePubMedGoogle Scholar
- Kadekoppala M, Holder AA: Merozoite surface proteins of the malaria parasite: the MSP1 complex and the MSP7 family. Int J Parasitol. 2010, 40: 1155-1161. 10.1016/j.ijpara.2010.04.008.View ArticlePubMedGoogle Scholar
- Kadekoppala M, Ogun SA, Howell S, Gunaratne RS, Holder AA: Systematic genetic analysis of the Plasmodium falciparum MSP7-like family reveals differences in protein expression, location, and importance in asexual growth of the blood-stage parasite. Eukaryot Cell. 2010, 9: 1064-1074. 10.1128/EC.00048-10.PubMed CentralView ArticlePubMedGoogle Scholar
- Kadekoppala M, O’Donnell RA, Grainger M, Crabb BS, Holder AA: Deletion of the Plasmodium falciparum merozoite surface protein 7 gene impairs parasite invasion of erythrocytes. Eukaryot Cell. 2008, 7: 2123-2132. 10.1128/EC.00274-08.PubMed CentralView ArticlePubMedGoogle Scholar
- Miller SK, Good RT, Drew DR, Delorenzi M, Sanders PR, Hodder AN, Speed TP, Cowman AF, de Koning-Ward TF, Crabb BS: A subset of Plasmodium falciparum SERA genes are expressed and appear to play an important role in the erythrocytic cycle. J Biol Chem. 2002, 277: 47524-47532. 10.1074/jbc.M206974200.View ArticlePubMedGoogle Scholar
- Hu G, Cabrera A, Kono M, Mok S, Chaal BK, Haase S, Engelberg K, Cheemadan S, Spielmann T, Preiser PR, Gilberger T-W, Bozdech Z: Transcriptional profiling of growth perturbations of the human malaria parasite Plasmodium falciparum. Nat Biotechnol. 2010, 28: 91-98. 10.1038/nbt.1597.View ArticlePubMedGoogle Scholar
- Haase S, Cabrera A, Langer C, Treeck M, Struck N, Herrmann S, Jansen PW, Bruchhaus I, Bachmann A, Dias S, Cowman AF, Stunnenberg HG, Spielmann T, Gilberger T-W: Characterization of a conserved rhoptry-associated leucine zipper-like protein in the malaria parasite Plasmodium falciparum. Infect Immun. 2008, 76: 879-887. 10.1128/IAI.00144-07.PubMed CentralView ArticlePubMedGoogle Scholar
- Taylor TE, Molyneux ME, Wirima JJ, Borgstein A, Goldring JD, Hommel M: Intravenous immunoglobulin in the treatment of paediatric cerebral malaria. Clin Exp Immunol. 1992, 90: 357-362.PubMed CentralView ArticlePubMedGoogle Scholar
- Bustamante LY, Bartholdson SJ, Crosnier C, Campos MG, Wanaguru M, Nguon C, Kwiatkowski DP, Wright GJ, Rayner JC: A full-length recombinant Plasmodium falciparum Pf RH5 protein induces inhibitory antibodies that are effective across common Pf RH5 genetic variants. Vaccine. 2013, 31: 373-379. 10.1016/j.vaccine.2012.10.106.PubMed CentralView ArticlePubMedGoogle Scholar
- Wanaguru M, Crosnier C, Johnson S, Rayner JC, Wright GJ: Biochemical analysis of the Plasmodium falciparum Erythrocyte-Binding Antigen-175 (EBA175)-Glycophorin-A interaction: implications for vaccine design. J Biol Chem. 2013, 288: 32106-32117. 10.1074/jbc.M113.484840.PubMed CentralView ArticlePubMedGoogle Scholar
- Bartholdson SJ, Bustamante LY, Crosnier C, Johnson S, Lea S, Rayner JC, Wright GJ: Semaphorin-7A is an erythrocyte receptor for P. falciparum merozoite-specific TRAP homolog, MTRAP. PLoS Pathog. 2012, 8: e1003031-10.1371/journal.ppat.1003031.PubMed CentralView ArticlePubMedGoogle Scholar
- Taechalertpaisarn T, Crosnier C, Bartholdson SJ, Hodder AN, Thompson J, Bustamante LY, Wilson DW, Sanders PR, Wright GJ, Rayner JC, Cowman AF, Gilson PR, Crabb BS: Biochemical and functional analysis of two Plasmodium falciparum blood-stage 6-cys proteins: P12 and P41. PLoS One. 2012, 7: e41937-10.1371/journal.pone.0041937.PubMed CentralView ArticlePubMedGoogle Scholar
- Crosnier C, Bustamante LY, Bartholdson SJ, Bei AK, Theron M, Uchikawa M, Mboup S, Ndir O, Kwiatkowski DP, Duraisingh MT, Rayner JC, Wright GJ: Basigin is a receptor essential for erythrocyte invasion by Plasmodium falciparum. Nature. 2011, 480: 534-537.PubMed CentralPubMedGoogle Scholar
- Muralidharan V, Oksman A, Pal P, Lindquist S, Goldberg DE: Plasmodium falciparum heat shock protein 110 stabilizes the asparagine repeat-rich parasite proteome during malarial fevers. Nat Commun. 2012, 3: 1310-PubMed CentralView ArticlePubMedGoogle Scholar
- Ntumngia FB, Bouyou-Akotet MK, Uhlemann A-C, Mordmüller B, Kremsner PG, Kun JFJ: Characterisation of a tryptophan-rich Plasmodium falciparum antigen associated with merozoites. Mol Biochem Parasitol. 2004, 137: 349-353. 10.1016/j.molbiopara.2004.06.008.View ArticlePubMedGoogle Scholar
- Chen L, Lopaticki S, Riglar DT, Dekiwadia C, Uboldi AD, Tham W-H, O’Neill MT, Richard D, Baum J, Ralph SA, Cowman AF: An EGF-like Protein Forms a Complex with Pf Rh5 and Is Required for Invasion of Human Erythrocytes by Plasmodium falciparum. PLoS Pathog. 2011, 7: e1002199-10.1371/journal.ppat.1002199.PubMed CentralView ArticlePubMedGoogle Scholar
- Douglas AD, Williams AR, Illingworth JJ, Kamuyu G, Biswas S, Goodman AL, Wyllie DH, Crosnier C, Miura K, Wright GJ, Long CA, Osier FH, Marsh K, Turner AV, Hill AVS, Draper SJ: The blood-stage malaria antigen Pf RH5 is susceptible to vaccine-inducible cross-strain neutralizing antibody. Nat Commun. 2011, 2 (May): 601-PubMed CentralView ArticlePubMedGoogle Scholar
- Hayton K, Gaur D, Liu A, Takahashi J, Henschen B, Singh S, Lambert L, Furuya T, Bouttenot R, Doll M, Nawaz F, Mu J, Jiang L, Miller LH, Wellems TE: Erythrocyte binding protein Pf RH5 polymorphisms determine species-specific pathways of Plasmodium falciparum invasion. Cell Host Microbe. 2008, 4: 40-51. 10.1016/j.chom.2008.06.001.PubMed CentralView ArticlePubMedGoogle Scholar
- Baum J, Chen L, Healer J, Lopaticki S, Boyle M, Triglia T, Ehlgen F, Ralph SA, Beeson JG, Cowman AF: Reticulocyte-binding protein homologue 5 - an essential adhesin involved in invasion of human erythrocytes by Plasmodium falciparum. Int J Parasitol. 2009, 39: 371-380. 10.1016/j.ijpara.2008.10.006.View ArticlePubMedGoogle Scholar
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