RESUMEN

Copper can exist in an oxidized and a reduced form, which enables the metal to play essential roles as a catalytic co-factor in redox reactions in many organisms. Copper confers redox activity to the terminal electron transport chain cytochrome c oxidase protein. Cytochrome c oxidase in yeast obtains copper for the CuB site in the Cox1 subunit from Cox11 in association with Cox19. When copper is chelated in growth medium, Plasmodium falciparum parasite development in infected red blood cells is inhibited and excess copper is toxic for the parasite. The gene of a 26 kDa Plasmodium falciparum PfCox19 protein with two Cx9C Cox19 copper binding motifs, was cloned and expressed as a 66 kDa fusion protein with maltose binding protein and affinity purified (rMBP-PfCox19). rMBP-PfCox19 bound copper measured by: a bicinchoninic acid release assay; an in vivo bacterial host growth inhibition assay; ascorbate oxidation inhibition and differential scanning fluorimetry. The native protein was detected by antibodies raised against rMBP-PfCox19. PfCox19 binds copper and is predicted to associate with PfCox11 in the insertion of copper into the CuB site of Plasmodium cytochrome c oxidase. Characterisation of the proteins involved in Plasmodium spp. copper metabolism will help us understand the role of cytochrome c oxidase and this essential metal in Plasmodium homeostasis.

Asunto(s)

Cobre , Plasmodium falciparum , Plasmodium falciparum/genética , Complejo IV de Transporte de Electrones/genética , Complejo IV de Transporte de Electrones/química

RESUMEN

BACKGROUND: Copper is an essential metal for living organisms as a catalytic co-factor for important enzymes, like cytochrome c oxidase the final enzyme in the electron transport chain. Plasmodium falciparum parasites in infected red blood cells are killed by excess copper and development in erythrocytes is inhibited by copper chelators. Cytochrome c oxidase in yeast obtains copper for the CuB site in the Cox1 subunit from Cox11. METHODS: A 162 amino acid carboxy-terminal domain of the P. falciparum Cox11 ortholog (PfCox11Ct) was recombinantly expressed and the rMBPPfCox11Ct affinity purified. Copper binding was measured in vitro and in Escherichia coli host cells. Site directed mutagenesis was used to identify key copper binding cysteines. Antibodies confirmed the expression of the native protein. RESULTS: rMBPPfCox11Ct was expressed as a 62 kDa protein fused with the maltose binding protein and affinity purified. rMBPPfCox11Ct bound copper measured by: a bicinchoninic acid release assay; atomic absorption spectroscopy; a bacterial host growth inhibition assay; ascorbate oxidation inhibition and in a thermal shift assay. The cysteine 157 amino acid was shown to be important for in vitro copper binding by PfCox11whilst Cys 60 was not. The native protein was detected by antibodies against rMBPPfCox11Ct. CONCLUSIONS: Plasmodium spp. express the PfCox11 protein which shares structural features and copper binding motifs with Cox11 from other species. PfCox11 binds copper and is, therefore, predicted to transfer copper to the CuB site of Plasmodium cytochrome c oxidase. Characterization of Plasmodium spp. proteins involved in copper metabolism will help sceintists understand the role of cytochrome c oxidase and this essential metal in Plasmodium homeostasis.

Asunto(s)

Complejo IV de Transporte de Electrones , Proteínas de Saccharomyces cerevisiae , Aminoácidos , Quelantes , Cobre/química , Cobre/metabolismo , Factor XI/metabolismo , Proteínas de la Membrana/metabolismo , Proteínas Mitocondriales , Plasmodium falciparum/metabolismo , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo

RESUMEN

Crocodile immunity has not been fully characterised with more studies on crocodile innate immunity than cell-mediated or humoral immunity. Crocodile immunoglobulin genes have been described but immunoglobulin proteins have not been isolated or studied biochemically. Two large proteins proposed to be crocodile IgM and IgY were isolated and purified from Crocodylus niloticus sera using two different protocols. A 50% (w/v) ammonium sulfate and a 15% (w/v) polyethylene glycol precipitation step was followed by Cibacron blue F3GA affinity- and Sephacryl-S300 gel filtration chromatography. An alternate purification protocol, with only two steps, involved thiophilic affinity- and Sephacryl-S300 gel filtration chromatography. The purified crocodile IgM resolved on reducing SDS-PAGE with an apparent mass of 180 kDa. Purified crocodile IgY resolved at 180 kDa alongside chicken IgY on a non-reducing SDS-PAGE gel, and is deduced to consist of two 66 kDa heavy and two 23 kDa light chains under reducing conditions. The thiophilic/gel filtration two-step protocol gave three-fold higher yields of isolated protein than the four-step precipitation/chromatography protocol. Antibodies against the isolated crocodile IgM and IgY were raised in chickens and affinity purified. The chicken antibodies differentiated between crocodile IgM and IgY and have the potential for use in the diagnosis of crocodile infections. The purified crocodile antibodies can be biochemically characterised and compared to mammalian and avian antibodies to give a better understanding of crocodile humoral immunity.

Asunto(s)

Caimanes y Cocodrilos/inmunología , Cromatografía de Afinidad/métodos , Cromatografía en Gel/métodos , Inmunoglobulina M/aislamiento & purificación , Inmunoglobulinas/aislamiento & purificación , Caimanes y Cocodrilos/sangre , Animales , Pollos , Cromatografía de Afinidad/instrumentación , Cromatografía en Gel/instrumentación , Inmunoglobulina M/sangre , Inmunoglobulina M/inmunología , Inmunoglobulinas/sangre , Inmunoglobulinas/inmunología , Sudáfrica

RESUMEN

Copper is an essential component of cuproproteins but can be toxic to cells, therefore copper metabolism is very carefully regulated within cells. To gain insight into trypanosome copper metabolism, Trypanosoma spp. genomic databases were screened for the presence of copper-containing and -transporting proteins. Among other genes encoding copper-binding proteins, a copper-transporting P-type ATPase (CuATPase) gene was identified. Sequence and phylogenetic analyses suggest that the gene codes for a Cu+ transporter belonging to the P1B-1 ATPase subfamily that has an N-terminal domain with copper binding motifs. The N-terminal cytosolic domains of the proteins from Trypanosoma congolense and Trypanosoma brucei brucei were recombinantly expressed in Escherichia coli as maltose binding protein (MBP) fusion proteins. These N-terminal domains bound copper in vitro and within E. coli cells, more than the control MBP fusion partner alone. The copper binding properties of the recombinant proteins were further confirmed when they inhibited copper catalysed ascorbate oxidation. Native CuATPases were detected in a western blot of lysates of T. congolense IL3000 and T. b. brucei ILTat1.1 bloodstream form parasites using affinity purified IgY antibodies against N-terminal domain peptides. The CuATPase was also detected by immunofluorescence in T. b. brucei bloodstream form parasites where it was associated with subcellular vesicles. In conclusion, Trypanosoma species express a copper-transporting P1B-1-type ATPase and together with other copper-binding proteins identified in the genomes of kinetoplastid parasites may constitute potential targets for anti-trypanosomal drug discovery.

Asunto(s)

ATPasas Transportadoras de Cobre , Cobre/metabolismo , Trypanosoma , Animales , Anticuerpos Antiprotozoarios/sangre , Antígenos de Protozoos/inmunología , Proteínas Portadoras/genética , Proteínas Portadoras/inmunología , Proteínas Portadoras/metabolismo , ATPasas Transportadoras de Cobre/química , ATPasas Transportadoras de Cobre/genética , ATPasas Transportadoras de Cobre/inmunología , ATPasas Transportadoras de Cobre/metabolismo , Vesículas Citoplasmáticas , Escherichia coli/genética , Transporte de Proteínas , Proteínas Recombinantes/genética , Trypanosoma/genética , Trypanosoma/metabolismo , Trypanosoma brucei brucei/genética , Trypanosoma brucei brucei/metabolismo , Trypanosoma congolense/genética , Trypanosoma congolense/metabolismo

RESUMEN

The intraerythrocytic malaria parasite digests haemoglobin to provide amino acids for metabolism and releases toxic haem that is sequestered into haemozoin, a non-toxic, insoluble, crystalline pigment. Following erythrocyte rupture, haemozoin is released into circulation and phagocytosed by monocytes. Phagocytosed haemozoin and antimalarial drugs have both been reported to modulate monocyte functions. This study determined the effects of therapeutic concentrations of seven antimalarial drugs; amodiaquine, artemisinin, chloroquine, doxycycline, primaquine, pyrimethamine and quinine, on the phagocytosis of ß-haematin (synthetic haemozoin) by two monocytic cell lines, J774A.1 and U937, and human peripheral blood mononuclear cells. A novel spectrophotometric method based on the absorbance (O.D 400 nm) of alkali/SDS treated monocytes containing ß-haematin was developed to complement counting phagocytosis with microscopy. The method has potential use for the large scale screening of monocyte phagocytic activity. Artemisinin, quinine, primaquine and pyrimethamine activated ß-haematin phagocytosis by 12% or more, whereas amodiaquine, chloroquine and doxycyline inhibited ß-haematin phagocytosis. In contrast, antimalarial drugs had minimal inhibitory effects on the phagocytosis of latex beads with only quinine resulting in more than 20% inhibition. Antimalarial drugs appear to alter monocyte phagocytic activity which has implications for the treatment, pathogenicity and adjunct therapies for malaria.

Asunto(s)

Antimaláricos/farmacología , Hemoproteínas/metabolismo , Monocitos/efectos de los fármacos , Fagocitosis/efectos de los fármacos , Amodiaquina/farmacología , Animales , Artemisininas/farmacología , Recuento de Células , Línea Celular , Cloroquina/farmacología , Doxiciclina/farmacología , Microanálisis por Sonda Electrónica , Hemo/análisis , Hemoproteínas/biosíntesis , Hemoproteínas/química , Hemoproteínas/ultraestructura , Humanos , Leucocitos Mononucleares/efectos de los fármacos , Leucocitos Mononucleares/metabolismo , Ratones , Microscopía Electrónica de Rastreo , Microscopía Electrónica de Transmisión , Monocitos/enzimología , Monocitos/metabolismo , Peroxidasa/aislamiento & purificación , Peroxidasa/metabolismo , Primaquina/farmacología , Pirimetamina/farmacología , Quinina/farmacología , Espectrofotometría , Temperatura , Células U937

RESUMEN

Coomassie brilliant blue R250, an anionic dye is the most popular stain to detect proteins resolved in SDS-PAGE gels. Crystal violet, a cationic dye was found to be versatile and stained proteins in SDS-PAGE gels and in zymograms. Stained proteins can be transferred to nitrocellulose and the stained proteins on the western detected with enzyme coupled antibodies. Staining can be reversed. Staining takes 3 h at RT or 30 min at 60 °C. Crystal violet stained some E. coli high and low molecular weight proteins not stained by Coomassie blue R250. Crystal violet stained down to 16 ng of protein, some five-fold lower than Coomassie blue, though the two stains had a similar linear dynamic range. The staining sensitivity could be increased to 2 ng when crystal violet and Coomassie blue were combined in a double staining/counterion dye formulation. The low concentrations of the dye without a destaining step reduces the costs of the technique and results in a more environmentally friendly stain compared to traditional staining methods.

Asunto(s)

Electroforesis en Gel de Poliacrilamida/métodos , Proteínas de Escherichia coli/química , Violeta de Genciana/química , Coloración y Etiquetado/métodos , Colorantes/química , Escherichia coli

RESUMEN

Measuring the concentration of proteins is an essential part of enzyme analysis or serves to monitor protein yields and losses during protein isolation procedures. Decisions on the usefulness of any protein isolation procedure depend on knowing the concentration of proteins before and after a procedure. Protein concentration in solution is generally measured with spectrophotometry in the UV range or in the presence of dyes or copper interacting with the protein. This review describes absorbance at 280 nm, the Lowry, Bradford (Coomassie Blue), and Smith (bicinchoninic acid) assays for measuring protein and includes suggestions for optimizing each method.

Asunto(s)

Indicadores y Reactivos/química , Proteínas/análisis , Quinolinas/química , Colorantes de Rosanilina/química , Espectrofotometría Ultravioleta

RESUMEN

In protein isolation, drug interaction studies, and proteomic or peptidomic procedures, protein solutions are often concentrated to remove solvents and undesirable molecules, to separate protein fractions, or to increase protein concentrations. Proteins can be concentrated by precipitation from solution with ammonium sulfate, polyethylene glycol, organic solvents, trichloroacetic acid, potassium chloride/sodium dodecyl sulfate thermal denaturation, and three-phase partitioning. Solvents can be removed by passage through a semipermeable barrier where protein solutions are forced against the barrier in a centrifuge tube or with increased pressure, concentrating proteins in the remaining solution. The semipermeable barrier can be surrounded by a hygroscopic reagent to draw the solvent across the membrane. Proteins can be concentrated by freeze-drying (lyophilization). Unique ligand interactions with proteins can be used to select for proteins by affinity purification or immunoprecipitation. All these methods to concentrate proteins are discussed.

Asunto(s)

Proteínas/química , Proteínas/aislamiento & purificación , Cromatografía de Afinidad , Interacciones Farmacológicas , Liofilización , Inmunoprecipitación , Péptidos/aislamiento & purificación , Polietilenglicoles , Proteómica , Dodecil Sulfato de Sodio/química , Ultrafiltración

RESUMEN

Sodium dodecyl sulfate polyacrylamide gel electrophoresis is a powerful tool to separate proteins according to their relative sizes. The technique provides information about both the size of a number of proteins and potentially the comparative amounts of each protein. To confirm the identity of proteins, proteins can be eluted from the gel and transferred electrophoretically to nitrocellulose for antibody-based detection. During electrophoresis, if the current is not stopped, proteins continue to pass down the gel and elute from the bottom of the gel. The standard electrophoresis gel apparatus can be employed with the addition of some tubing and alterations to the separating gel to collect proteins separated by size as they elute from the base of the gel as described in this chapter. Complex protein mixtures can be separated into multiple fractions containing single proteins in a few hours. Small amounts (<500 µg of protein) of protein sample can be fractionated.

Asunto(s)

Electroforesis en Gel de Poliacrilamida/instrumentación , Proteínas/aislamiento & purificación , Resinas Acrílicas/química , Animales , Colodión/química , Mezclas Complejas/química , Electroforesis en Gel de Poliacrilamida/métodos , Diseño de Equipo , Escherichia coli/química , Proteínas de Escherichia coli/aislamiento & purificación , Geles/química , Humanos , Indicadores y Reactivos , Peso Molecular , Tamaño de la Muestra , Dodecil Sulfato de Sodio/química

RESUMEN

After SDS-polyacrylamide gel electrophoresis proteins are "fixed" in the gel to prevent dispersion of the proteins and visualized by staining with a chromogenic dye. Dyes like Coomassie Blue R-250, Amido Black, and Direct Red 81 are usually dissolved in an acetic acid-methanol-water mixture. During staining the dye solvent mixture infuses the gel and interacts with the protein. Acetic acid and methanol denature the protein and provide an acidic environment enhancing the interactions with dyes. After staining, the dye that is in the gel and not bound to the protein, is removed using the solvent medium the dyes were dissolved in. Over 2-3 h the solution surrounding the gel becomes colored, the gel becomes lighter and the protein bands remain dark and the contrast against the surrounding gel improves. This chapter describes how each of the individual components in the dye solution interact with the protein resulting in a stained protein band in a clear SDS-polyacrylamide electrophoresis gel.

Asunto(s)

Ácido Acético , Electroforesis en Gel de Poliacrilamida , Metanol , Proteínas , Electroforesis en Gel de Poliacrilamida/métodos , Proteínas/química , Coloración y Etiquetado

RESUMEN

Proteins separated by SDS-polyacrylamide gel electrophoresis need to be stained with organic dyes to be visualized. Once stained the intensity of each stained protein band can be used to compare the differences in protein concentration and to measure the relative concentration of any protein band. The most popular standard protein staining is with Coomassie Blue R-250 which takes an hour to stain proteins to saturation and several hours to remove background staining. Direct Red 81 and Amido Black stain proteins within 2.5 min and staining is complete by 10 min. Here the rapid staining of proteins with Direct Red 81 and Amido Black in comparison to staining with Coomassie Blue R-250 is described.

Asunto(s)

Negro de Almidón , Compuestos Azo , Electroforesis en Gel de Poliacrilamida , Proteínas , Coloración y Etiquetado , Proteínas/análisis , Colorantes de Rosanilina , Coloración y Etiquetado/métodos

RESUMEN

BACKGROUND: Early detection is crucial for the effective treatment of malaria, particularly in those cases infected with Plasmodium falciparum. There is a need for diagnostic devices with the capacity to distinguish P. falciparum from other strains of malaria. Here, aptamers generated against targeted species-specific epitopes of P. falciparum lactate dehydrogenase (rPfLDH) are described. RESULTS: Two classes of aptamers bearing high binding affinity and specificity for recombinant P. falciparum lactate dehydrogenase (rPfLDH) and P. falciparum-specific lactate dehydrogenase epitopic oligopeptide (LDHp) were separately generated. Structurally-relevant moieties with particular consensus sequences (GGTAG and GGCG) were found in aptamers reported here and previously published, confirming their importance in recognition of the target, while novel moieties particular to this work (ATTAT and poly-A stretches) were identified. Aptamers with diagnostically-supportive functions were synthesized, prime examples of which are the aptamers designated as LDHp 1, LDHp 11 and rLDH 4 and rLDH 15 in work presented herein. Of the sampled aptamers raised against the recombinant protein, rLDH 4 showed the highest binding to the target rPfLDH in the ELONA assay, with both rLDH 4 and rLDH 15 indicating an ability to discriminate between rPfLDH and rPvLDH. LDHp 11 was generated against a peptide selected as a unique P. falciparum LDH peptide. The aptamer, LDHp 11, like antibodies against the same peptide, only detected rPfLDH and discriminated between rPfLDH and rPvLDH. This was supported by affinity binding experiments where only aptamers generated against a unique species-specific epitope showed an ability to preferentially bind to rPfLDH relative to rPvLDH rather than those generated against the whole recombinant protein. In addition, rLDH 4 and LDHp 11 demonstrated in situ binding to P. falciparum cells during confocal microscopy. CONCLUSIONS: The utilization and application of LDHp 11, an aptamer generated against a unique species-specific epitope of P. falciparum LDH indicated the ability to discriminate between recombinant P. falciparum and Plasmodium vivax LDH. This aptamer holds promise as a biorecognition element in malaria diagnostic devices for the detection, and differentiation, of P. falciparum and P. vivax malaria infections. This study paves the way to explore aptamer generation against targeted species-specific epitopes of other Plasmodium species.

Asunto(s)

Aptámeros de Péptidos/metabolismo , Epítopos/metabolismo , L-Lactato Deshidrogenasa/metabolismo , Plasmodium falciparum/enzimología , Proteínas Protozoarias/metabolismo , Proteínas Recombinantes/metabolismo

RESUMEN

BACKGROUND: Plasmodium knowlesi is recognised as the main cause of human malaria in Southeast Asia. The disease is often misdiagnosed as P. falciparum or P. malariae infections by microscopy, and the disease is difficult to eliminate due to its presence in both humans and monkeys. P. knowlesi infections can rapidly cause severe disease and require prompt diagnosis and treatment. No protein biomarker exists for the rapid diagnostic test (RDT) detection of P. knowlesi infections. Plasmodium knowlesi infections can be diagnosed by PCR. METHODS AND PRINCIPAL FINDINGS: Phosphoethanolamine-N-methyltransferase (PMT) is involved in malaria lipid biosynthesis and is not found in the human host. The P. falciparum, P. vivax and P. knowlesi PMT proteins were recombinantly expressed in BL21(DE3) Escherichia coli host cells, affinity purified and used to raise antibodies in chickens. Antibodies against each recombinant PMT protein all detected all three recombinant proteins and the native 29 kDa P. falciparum PMT protein on western blots and in ELISA. Antibodies against a PMT epitope (PLENNQYTDEGVKC) common to all three PMT orthologues detected all three proteins. Antibodies against unique peptides from each orthologue of PMT, PfCEVEHKYLHENKE, PvVYSIKEYNSLKDC, PkLYPTDEYNSLKDC detected only the parent protein in western blots and P. falciparum infected red blood cell lysates or blood lysates spiked with the respective proteins. Similar concentrations of PfPMT and the control, PfLDH, were detected in the same parasite lysate. The recombinant PfPMT protein was detected by a human anti-malaria antibody pool. CONCLUSION: PMT, like the pan-specific LDH biomarker used in RDT tests, is both soluble, present at comparable concentrations in the parasite and constitutes a promising antimalarial drug target. PMT is absent from the human proteome. PMT has the potential as a biomarker for human malaria and in particular as the first P. knowlesi specific protein with diagnostic potential for the identification of a P. knowlesi infection.

Asunto(s)

Malaria/metabolismo , Fosfatidiletanolamina N-Metiltransferasa/metabolismo , Plasmodium falciparum , Plasmodium knowlesi , Animales , Biomarcadores/metabolismo , Western Blotting , Pollos , Biología Computacional , Simulación por Computador , Electroforesis en Gel de Poliacrilamida , Ensayo de Inmunoadsorción Enzimática , Escherichia coli , Humanos , Inmunoglobulina G/metabolismo , Inmunoglobulinas/metabolismo , Fosfatidiletanolamina N-Metiltransferasa/genética , Plasmodium falciparum/genética , Plasmodium falciparum/metabolismo , Plasmodium knowlesi/genética , Plasmodium knowlesi/metabolismo , Proteínas Recombinantes/metabolismo , Alineación de Secuencia

RESUMEN

Malaria rapid diagnostic tests (RDTs) are immunochromatographic tests detecting Plasmodial histidine-rich protein 2 (HRP2), lactate dehydrogenase (LDH) and aldolase. HRP2 is only expressed by Plasmodium falciparum parasites and the protein is not expressed in several geographic isolates. LDH-based tests lack sensitivity compared to HRP2 tests. This study explored the potential of the Plasmodial glycolytic enzyme, glyceraldehyde-3-phosphate dehydrogenase (GAPDH), as a new malaria diagnostic biomarker. The P. falciparum and P. yoelii proteins were recombinantly expressed in BL21(DE3) Escherischia coli host cells and affinity purified. Two epitopes (CADGFLLIGEKKVSVFA and CAEKDPSQIPWGKCQV) specific to P. falciparum GAPDH and one common to all mammalian malaria species (CKDDTPIYVMGINH) were identified. Antibodies were raised in chickens against the two recombinant proteins and the three epitopes and affinity purified. The antibodies detected the native protein in parasite lysates as a 38 kDa protein and immunofluorescence verified a parasite cytosolic localization for the native protein. The antibodies suggested a 4-6 fold higher concentration of native PfGAPDH compared to PfLDH in immunoprecipitation and ELISA formats, consistent with published proteomic data. PfGAPDH shows interesting potential as a malaria diagnostic biomarker.

Asunto(s)

Anticuerpos Antiprotozoarios/inmunología , Gliceraldehído-3-Fosfato Deshidrogenasa (Fosforilante)/metabolismo , Malaria Falciparum/diagnóstico , Plasmodium falciparum/enzimología , Secuencia de Aminoácidos , Animales , Anticuerpos Antiprotozoarios/biosíntesis , Especificidad de Anticuerpos , Antígenos de Protozoos/aislamiento & purificación , Biomarcadores/análisis , Western Blotting , Pollos , Cromatografía de Afinidad , Cromatografía en Gel , Diagnóstico Diferencial , Ensayo de Inmunoadsorción Enzimática , Epítopos/inmunología , Epítopos/aislamiento & purificación , Técnica del Anticuerpo Fluorescente , Fructosa-Bifosfato Aldolasa/aislamiento & purificación , Gliceraldehído-3-Fosfato Deshidrogenasa (Fosforilante)/química , Gliceraldehído-3-Fosfato Deshidrogenasa (Fosforilante)/inmunología , Inmunoglobulina G/biosíntesis , Inmunoglobulina G/inmunología , Inmunoglobulina G/aislamiento & purificación , Inmunoglobulinas/inmunología , Inmunoprecipitación , L-Lactato Deshidrogenasa/inmunología , L-Lactato Deshidrogenasa/aislamiento & purificación , L-Lactato Deshidrogenasa/metabolismo , Plasmodium falciparum/inmunología , Plasmodium yoelii/enzimología , Plasmodium yoelii/inmunología , Proteínas Protozoarias/aislamiento & purificación , Conejos , Proteínas Recombinantes/inmunología , Proteínas Recombinantes/aislamiento & purificación , Proteínas Recombinantes/metabolismo

RESUMEN

Pheroid® technology was assessed as an alternative to Freund's adjuvant to raise antibodies in experimental animals. Chickens were immunized with two recombinantly expressed Plasmodium falciparum proteins, lactate dehydrogenase (PfLDH) and glyceraldehyde-3-phosphate dehydrogenase (PfGAPDH), alone or in combination with Freund's adjuvant or Pheroid®. Chicken egg yolk antibodies (IgY) were isolated and compared for specificity, sensitivity and yield. Freund's adjuvant and Pheroid® stimulated prolonged antibody responses in chickens against both antigens. Affinity purified antibodies had specificity for the recombinant and the native proteins on Western blots. Antibodies generated in the presence of Freund's adjuvant had high sensitivity for both antigens. Pheroid® generated antibodies that detected the lowest concentration of recombinant PfLDH. Freund's adjuvant and Pheroid® both improved chicken IgY yields, with Pheroid® showing a 2-fold increase relative to controls. Pheroid® was well-tolerated in chickens and has potential for development as a safe adjuvant for testing alternative stimulatory factors to improve adjuvant formulations.

Asunto(s)

Anticuerpos Antiprotozoarios/inmunología , Formación de Anticuerpos , Antígenos de Protozoos/inmunología , Adyuvante de Freund , Gliceraldehído-3-Fosfato Deshidrogenasa (Fosforilante)/inmunología , L-Lactato Deshidrogenasa/inmunología , Plasmodium falciparum/inmunología , Secuencia de Aminoácidos , Animales , Especificidad de Anticuerpos/inmunología , Pollos , Gliceraldehído-3-Fosfato Deshidrogenasa (Fosforilante)/química , Inmunización , Inmunoglobulinas/inmunología , L-Lactato Deshidrogenasa/química , Malaria Falciparum , Datos de Secuencia Molecular , Proteínas Recombinantes/inmunología , Alineación de Secuencia

RESUMEN

In protein isolation, proteomic, or peptidomic procedures, protein solutions are often concentrated to remove solvents and undesirable molecules, to separate protein fractions or to increase protein concentrations. Proteins can be concentrated by precipitation from solution with ammonium sulfate, polyethylene glycol, organic solvent, trichloroacetic acid, potassium chloride/sodium dodecyl sulfate, and three-phase partitioning. Solvents can be removed by passage through a semipermeable barrier where protein solutions are forced against the barrier in a centrifuge tube or with increased pressure concentrating protein in the remaining solution. The semipermeable barrier can be surrounded by a hygroscopic reagent to draw the solvent across the membrane. Proteins can be concentrated by freeze-drying (lyophilization). All these methods to concentrate proteins are discussed.

Asunto(s)

Proteínas/aislamiento & purificación , Adsorción , Sulfato de Amonio/química , Animales , Centrifugación/métodos , Precipitación Química , Diálisis/métodos , Electroforesis en Gel de Poliacrilamida/métodos , Liofilización/métodos , Humanos , Polietilenglicoles , Proteómica/métodos , Dodecil Sulfato de Sodio/química , Solventes/química , Ácido Tricloroacético/química , Ultrafiltración/métodos

RESUMEN

Proteins bind to nitrocellulose membranes when applied directly or after electrophoretic transfer from polyacrylamide electrophoresis gels. Proteins can be stained for visualization with organic dyes Ponceau S, amido black, Coomassie Blue, and colloidal silver/gold and the intensity of the stain is directly proportional to the amount of protein present. Chemicals that interfere with dye/protein interactions in solution can be removed by washing the nitrocellulose after protein application. A method is described whereby protein-dye complexes attached to the nitrocellulose can be solubilized, dissolving the nitrocellulose and releasing dye into solution for detection by a spectrophotometer. The concentration of the dyes Ponceau S, amido black, and colloidal silver is proportional to the concentration of protein. Proteins transferred electrophoretically from SDS-PAGE, isoelectric focusing, or 2D gels to nitrocellulose can be stained with amido black, protein bands excised, and the bound dye detected in a spectrophotometer to quantify proteins in the individual protein bands.

Asunto(s)

Colodión/química , Colorantes/química , Proteínas/aislamiento & purificación , Coloración y Etiquetado/métodos , Negro de Almidón/química , Animales , Compuestos Azo/química , Western Blotting/métodos , Electroforesis en Gel de Poliacrilamida/métodos , Oro Coloide/química , Humanos , Focalización Isoeléctrica/métodos , Membranas Artificiales , Proteínas/análisis , Colorantes de Rosanilina/química , Plata/química , Espectrofotometría/métodos

RESUMEN

Measuring the concentration of proteins is an essential part of enzyme evaluations or to monitor protein yields during protein isolation procedures. Decisions on the usefulness of any isolation procedure depend on knowing the relative concentrations of a particular protein or enzyme in relation to the concentrations of all the proteins present. Protein concentration in solution is generally measured with spectrophotometry in the UV range or in the presence of dyes or copper interacting with the protein. This review describes protein determination methods for measuring protein concentration in solution.

Asunto(s)

Proteínas/análisis , Espectrofotometría/métodos , Proteínas/química , Quinolinas/química , Colorantes de Rosanilina/química

RESUMEN

Copper is an essential micronutrient for all living organisms as an important catalytic co-factor for key enzymes. In higher eukaryotes intracellular copper is distributed by copper metallochaperones. Copper chelators such as neocuproine and tetrathiomolybdate inhibit Plasmodium falciparum erythrocytic development, indicating a requirement for copper by the parasite. A screen of the P. falciparum genome database identified eight potential copper-requiring protein orthologs, including four candidate copper metallochaperones implicated in the delivery of copper to cytochrome-c oxidase. A P. falciparum Cox17 ortholog (PfCox17) was recombinantly expressed and the purified protein bound reduced copper in vitro. PfCox17 was localised to the parasite cytoplasm. Characterisation of plasmodial proteins involved in copper metabolism will help us understand the role of this essential microelement in plasmodial homeostasis.

Asunto(s)

Proteínas Portadoras/aislamiento & purificación , Cobre/metabolismo , Metalochaperonas/aislamiento & purificación , Plasmodium falciparum/química , Secuencia de Aminoácidos , Animales , Ácido Ascórbico/metabolismo , Proteínas Portadoras/química , Pollos , Análisis por Conglomerados , Femenino , Humanos , Metalochaperonas/química , Conformación Molecular , Datos de Secuencia Molecular , Plasmodium falciparum/genética , Conejos , Proteínas Recombinantes/química , Proteínas Recombinantes/aislamiento & purificación , Alineación de Secuencia , Homología de Secuencia de Aminoácido

RESUMEN

In the last decade, there has been an upsurge of interest in developing malaria rapid diagnostic test (RDT) kits for the detection of Plasmodium species. Three antigens - Plasmodium falciparum histidine-rich protein 2 (PfHRP2), plasmodial aldolase and plasmodial lactate dehydrogenase (pLDH) - are currently used for RDTs. Tests targeting HRP2 contribute to more than 90% of the malaria RDTs in current use. However, the specificities, sensitivities, numbers of false positives, numbers of false negatives and temperature tolerances of these tests vary considerably, illustrating the difficulties and challenges facing current RDTs. This paper describes recent developments in malaria RDTs, reviewing RDTs detecting PfHRP2, pLDH and plasmodial aldolase. The difficulties associated with RDTs, such as genetic variability in the Pfhrp2 gene and the persistence of antigens in the bloodstream following the elimination of parasites, are discussed. The prospect of overcoming the problems associated with current RDTs with a new generation of alternative malaria antigen targets is also described.

Asunto(s)

Pruebas Diagnósticas de Rutina/métodos , Malaria/diagnóstico , Plasmodium/aislamiento & purificación , Sistemas de Atención de Punto , Antígenos de Protozoos/análisis , Humanos , Inmunoensayo/métodos , Sensibilidad y Especificidad