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1.
Protein Sci ; 28(4): 800-807, 2019 04.
Artículo en Inglés | MEDLINE | ID: mdl-30653276

RESUMEN

The production of recombinant proteins in bacteria has increased significantly in recent years, becoming a common tool for both research and the industrial production of proteins. One of the requirements of this methodology is to obtain the desired protein without contaminants. However, this goal cannot always be readily achieved. Multiple strategies have been developed to improve the quality of the desired protein product. Nevertheless, contamination with molecular chaperones is one of the recalcitrant problems that still affects the quality of the obtained proteins. The ability of chaperones to bind to unfolded proteins or to regions where the polypeptide chain is exposed make the removal of the contamination during purification challenging to achieve. This work aimed to develop a strategy to remove contaminating DnaK, one of the homologous Hsp70 molecular chaperones found in Escherichia coli, from purified recombinant proteins. For this purpose, we developed a methodology that captures the DnaK from the contaminating proteins by co-incubation with a GST-cleanser protein that has free functional binding sites for the chaperone. The cleanser protein can then be easily removed together with the captured DnaK. Here, we demonstrated the utility of our system by decontaminating a Histidine-tagged recombinant protein in a batch process. The addition of the GST-cleanser protein in the presence of ATP-Mg eliminates the DnaK contamination substantially. Thus, our decontaminant strategy results versatile and straightforward and can be applied to proteins obtained with different expression and purifications systems as well as to small samples or large volume preparations.


Asunto(s)
Proteínas de Escherichia coli/aislamiento & purificación , Escherichia coli/química , Proteínas HSP70 de Choque Térmico/aislamiento & purificación , Proteínas Recombinantes/química , Sitios de Unión , Western Blotting , Electroforesis en Gel de Poliacrilamida , Proteínas Inmovilizadas/química
2.
Genet Mol Res ; 16(1)2017 Mar 15.
Artículo en Inglés | MEDLINE | ID: mdl-28301667

RESUMEN

Members of the Eimeria genus are protozoan parasites of the subphylum Apicomplexa (Eimeriidae family), and belong to the coccidia group. Eimeria tenella is one of the most pathogenic species owing to its ability to penetrate the mucosa, and cause inflammation and damage. It is an obligate intracellular parasite that causes disease by destroying the host cells during multiplication. Heat shock protein 70 (HSP70) is a molecular chaperone that prevents cellular stress. The objective of this study was to clone, sequence, and express E. tenella HSP70 protein. After selecting the region of highest hydrophilicity in the hsp70 gene, we cloned complementary DNA (cDNA) into a pTrcHis2-TOPO vector and transformed it into TOP10 Escherichia coli cells; after induction, the bacteria expressed a 23-kDa protein with insoluble expression levels of approximately 5 mg/L. In summary, the partial hsp70 gene was successfully expressed in E. coli, producing a 23-kDa protein under insoluble conditions, and the antigen characteristics predicted by hydrophilicity analysis suggest the development of a vaccine for use in avian coccidiosis.


Asunto(s)
Eimeria tenella/genética , Proteínas HSP70 de Choque Térmico/genética , Proteínas Protozoarias/genética , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Pollos , Escherichia coli , Expresión Génica , Proteínas HSP70 de Choque Térmico/biosíntesis , Proteínas HSP70 de Choque Térmico/química , Proteínas HSP70 de Choque Térmico/aislamiento & purificación , Interacciones Hidrofóbicas e Hidrofílicas , Proteínas Protozoarias/biosíntesis , Proteínas Protozoarias/química , Proteínas Protozoarias/aislamiento & purificación , Análisis de Secuencia de ADN
3.
Acta Trop ; 122(2): 169-76, 2012 May.
Artículo en Inglés | MEDLINE | ID: mdl-22266120

RESUMEN

A 30-kDa surface collagen binding protein peroxiredoxin of Entamoeba histolytica (EhCBP30) was evaluated either alone or fused to the chaperone (CHP) or ATPase (ATP) domains of heat shock protein 70 of Trypanosoma cruzi (TcHSP70) as a vaccine candidate in a hamster model of experimental amoebic liver abscess (ALA) development. Three constructs were produced containing the EhCBP30 DNA sequence, one expressing EhCBP30 and two expressing EhCBP30 fused to either CHP or ATP domains of TcHSP70. High purity recombinant proteins rEhCBP30, rEhCBP30-CHP and rEhCBP30-ATP with N-terminal His tag were obtained by single step affinity purification. Hamsters were immunized without adjuvant with the antigenic recombinant proteins and then challenged intrahepatically with E. histolytica trophozoites. A 70% decrease in ALA development was detected in hamsters immunized with rEhCBP30 and rEhCBP30-CHP, while animals immunized with rEhCBP30-ATP did not show a statistically significant decrease in ALA formation compared with non-immunized animals. Histological analysis of liver tissue showed that the inflammatory infiltrate was discrete or moderate in hamsters immunized with rEhCBP30 or rEhCBP30-CHP compared with that observed in control hamsters or hamsters immunized with rEhCBP30-ATP. These results suggest that rEhCBP30 and rEhCBP30-CHP are able to induce an effective immune response that may protect hamsters against ALA development.


Asunto(s)
Anticuerpos Antiprotozoarios/sangre , Antígenos de Protozoos/inmunología , Entamoeba histolytica/inmunología , Absceso Hepático Amebiano/prevención & control , Vacunas Antiprotozoos/inmunología , Animales , Antígenos de Protozoos/aislamiento & purificación , Clonación Molecular , Cricetinae , Entamoeba histolytica/genética , Femenino , Proteínas HSP70 de Choque Térmico/inmunología , Proteínas HSP70 de Choque Térmico/aislamiento & purificación , Inmunización , Hígado/patología , Absceso Hepático Amebiano/parasitología , Masculino , Mesocricetus , Ratones , Ratones Endogámicos BALB C , Modelos Animales , Proteínas Protozoarias/inmunología , Proteínas Protozoarias/aislamiento & purificación , Proteínas Recombinantes/inmunología , Proteínas Recombinantes/aislamiento & purificación , Trofozoítos , Trypanosoma cruzi/genética
4.
Plant Cell Physiol ; 46(6): 997-1006, 2005 Jun.
Artículo en Inglés | MEDLINE | ID: mdl-15840644

RESUMEN

Different preparations of antibodies against 62 kDa NADP-malic enzyme (NADP-ME) from purified maize leaves cross-react with a 72 kDa protein from diverse tissues in many species. A 72 kDa protein, suggested to be a non-photosynthetic NADP-ME, has been purified from several plant species. However, to date, a cDNA coding for this putative 72 kDa NADP-ME has not been isolated. The screening of maize and tobacco leaf expression libraries using antibodies against purified 62 kDa NADP-ME allowed the identification of a heat shock protein (Hsp70). In addition, tandem mass spectrometry (MS/MS) studies indicate that along with NADP-ME, a 72 kDa protein, identified as an Hsp70 and reacting with the antibodies, is also purified from maize roots. On the other hand, the screening of a maize root cDNA library revealed the existence of a cDNA that encodes a mature 66 kDa NADP-ME. These results suggest that the 72 kDa protein is not actually an NADP-ME but in fact an Hsp70, at least in maize and tobacco. Probably, NADP-ME-Hsp70 association, taking place at least when preparing crude extracts, can lead to a co-purification of the proteins and can thus explain the cross-reaction of the antibodies. In the present work, we analyse and discuss a probable interaction of NADP-ME with Hsp70.


Asunto(s)
Proteínas HSP70 de Choque Térmico/aislamiento & purificación , Malato Deshidrogenasa/aislamiento & purificación , Proteínas de Plantas/aislamiento & purificación , Secuencia de Aminoácidos , Secuencia de Bases , Reacciones Cruzadas , ADN Complementario/genética , ADN de Plantas/genética , Estabilidad de Enzimas , Biblioteca de Genes , Proteínas Fluorescentes Verdes/genética , Proteínas Fluorescentes Verdes/metabolismo , Proteínas HSP70 de Choque Térmico/genética , Proteínas HSP70 de Choque Térmico/inmunología , Proteínas HSP70 de Choque Térmico/metabolismo , Cinética , Malato Deshidrogenasa/genética , Malato Deshidrogenasa/inmunología , Malato Deshidrogenasa/metabolismo , Datos de Secuencia Molecular , Proteínas de Plantas/genética , Proteínas de Plantas/inmunología , Proteínas de Plantas/metabolismo , Plantas Modificadas Genéticamente , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/aislamiento & purificación , Proteínas Recombinantes de Fusión/metabolismo , Nicotiana/genética , Nicotiana/metabolismo , Zea mays/genética , Zea mays/metabolismo
5.
Protein Expr Purif ; 25(3): 503-7, 2002 Aug.
Artículo en Inglés | MEDLINE | ID: mdl-12182832

RESUMEN

The use of fusion proteins for recombinant protein expression in Escherichia coli has become popular because the carrier increases protein solubility, standardizes expression levels, and facilitates purification of the fusion products. However, we have observed that the peptide regions that fuse the carrier to the protein of interest bind E. coli Hsp70 molecular chaperones (DnaK) depending on their amino acid composition, resulting in an unwanted contamination during protein purification. Here we describe an approach that helps to circumvent this unwanted contamination. First, the appropriate amino acids surrounding and comprising the cloning site are chosen by using a software based on an algorithm already developed to decrease to a minimum the propensity of the fusion protein to bind DnaK. Second, DnaK contamination is significantly reduced by washing the fusion protein bound to the purification resin with MgATP plus soluble denatured E. coli proteins before elution. The approach can also be applied to eliminate other molecular chaperones.


Asunto(s)
Proteínas Bacterianas , Escherichia coli/química , Proteínas HSP70 de Choque Térmico/aislamiento & purificación , Proteínas Recombinantes de Fusión/aislamiento & purificación , Secuencia de Aminoácidos , Escherichia coli/genética , Proteínas de Escherichia coli/análisis , Proteínas de Escherichia coli/aislamiento & purificación , Proteínas HSP70 de Choque Térmico/análisis , Datos de Secuencia Molecular , Proteínas Recombinantes de Fusión/genética , Programas Informáticos
6.
J Clin Microbiol ; 40(2): 359-65, 2002 Feb.
Artículo en Inglés | MEDLINE | ID: mdl-11825942

RESUMEN

The 87-kDa antigen derived from the fungal pathogen Paracoccidioides brasiliensis can be detected in the sera of infected patients, and its levels have been shown to correlate well with response to treatment and with clinical cure. Despite its potential importance, the antigen has been poorly characterized. The 87-kDa antigen was purified to homogeneity via preparative gel electrophoresis; N-terminal amino acid sequencing revealed substantial homology with heat shock proteins (hsps) from a variety of organisms. A monoclonal antibody (MAb) raised against a Histoplasma capsulatum 80-kDa hsp showed cross-reactivity to the purified 87-kDa antigen via Western blotting, and the 87-kDa-specific MAb P1B demonstrated that the antigen was expressed at higher levels in yeast than in mycelia by the same technique. Enzyme-linked immunosorbent assay and immunofluorescence reactivity using P1B confirmed increased expression of the 87-kDa antigen during the temperature-induced transformation of mycelia to yeast. Yeast-to-mycelium transformation was accompanied by a fall in expression, although the 87-kDa antigen was clearly constitutively expressed in both phases. Immunochemical staining of tissues from patients with MAb P1B who were infected with P. brasiliensis confirmed in vivo expression of the 87-kDa antigen by yeasts, and identification of this antigen via this method appears to be a useful adjunct to other methods used to diagnose paracoccidioidomycosis.


Asunto(s)
Antígenos Fúngicos , Proteínas HSP70 de Choque Térmico , Paracoccidioides/inmunología , Paracoccidioidomicosis/diagnóstico , Secuencia de Aminoácidos , Antígenos Fúngicos/análisis , Antígenos Fúngicos/química , Antígenos Fúngicos/aislamiento & purificación , Biopsia , Western Blotting , Proteínas HSP70 de Choque Térmico/análisis , Proteínas HSP70 de Choque Térmico/química , Proteínas HSP70 de Choque Térmico/inmunología , Proteínas HSP70 de Choque Térmico/aislamiento & purificación , Humanos , Inmunohistoquímica , Datos de Secuencia Molecular , Paracoccidioides/crecimiento & desarrollo , Paracoccidioidomicosis/microbiología
7.
Cell Stress Chaperones ; 3(1): 67-77, 1998 Mar.
Artículo en Inglés | MEDLINE | ID: mdl-9585183

RESUMEN

The presence of antibodies against the major stress protein, Hsp70, in patients with autoimmune diseases led us to hypothesize that Hsp70 may occur extracellularly, and could exert chaperoning and regulatory effects on various cells. We examined the action of pure Hsp/Hsc70 on the main physiological functions of human promonocytic U-937 cells. The protein was isolated from calf muscle and was shown to be a mixture of inducible Hsp70 (60%) and constitutive Hsc70 (40%) isoforms. It was observed that the addition of the protein up-regulated two major monocyte/macrophage differentiation markers, CD11c and CD23, by 20-35%, while it had no effect on CD14. The experiments performed to investigate the influence of Hsp/Hsc70 on the reaction of U-937 cells to differentiation stimuli demonstrated that the addition of the protein prior to PMA was able to inhibit binding of proper transcription factors to double-symmetry and cAMP-response elements of the c-fos early response gene promoter. Administration of exogenous Hsp/Hsc70 prior to treatment with the tumor necrosis factor-alpha significantly lowered the number of apoptotic and necrotic cells. In no case did the control protein, ovalbumin, taken in the same concentration give a comparable effect on U-937 cells. Since the Hsp/Hsc70 effects occurred within the first hour of co-incubation, and therefore they might be explained by its interaction with the cell surface, we assayed binding of the biotinylated protein to U-937 cells by immunoenzyme assay, flow cytometry and indirect immunofluorescence. Using these three techniques we were able to detect Hsp/Hsc70 bound to cells after a 20 min incubation. According to flow cytometry data, at this time 32% of cells were positively stained with streptavidin-FITC. Immunofluorescence studies demonstrated Hsp/Hsc70 bound to the cell surface after a 20 min incubation followed by induction of patch and cap-like structures. One hour later, the majority of the protein had been internalized by U-937 cells.


Asunto(s)
Proteínas Portadoras/farmacología , Proteínas HSP70 de Choque Térmico/farmacología , Monocitos/citología , Animales , Antígenos CD/análisis , Apoptosis , Proteínas Portadoras/aislamiento & purificación , Proteínas Portadoras/metabolismo , Bovinos , Diferenciación Celular , División Celular , Línea Celular , ADN/metabolismo , Endocitosis , Citometría de Flujo , Genes fos/genética , Proteínas del Choque Térmico HSC70 , Proteínas HSP70 de Choque Térmico/aislamiento & purificación , Proteínas HSP70 de Choque Térmico/metabolismo , Humanos , Monocitos/metabolismo , Músculo Esquelético , Regiones Promotoras Genéticas/genética , Acetato de Tetradecanoilforbol/farmacología , Factores de Transcripción/metabolismo , Factor de Necrosis Tumoral alfa/toxicidad
8.
Biol Res ; 26(1-2): 313-4, 1993.
Artículo en Inglés | MEDLINE | ID: mdl-7670543

RESUMEN

To identify the members of the HSP70 and HSP60 families of Trypanosoma cruzi, we analysed 35S methionine epimastigote cells by two dimensional Western blot. At 29 degrees C, an HSP70 monoclonal antibody (anti-D. melanogaster) recognized eight isotypes. At least five of these were heat-induced. Polyclonal antibody against the 65 KDa antigen (anti-M. tuberculosis) recognized three isotypes with identical molecular weights, but different microliters. Only one isoform was heat induced. The cellular distribution of HSP70 and HSP60 was studied by immunoelectron microscopy. Anti-HSP70 reactive protein was localized in the cytoplasm, mitochondria and nucleus, while anti-HSP60 protein was found in the mitochondrion and in close association with the kinetoplast. To characterize the HSP60 gene and its proteins, we isolated a genomic T. cruzi clone encoding the HSP60 gene. T. cruzi HSP60 genes could be shown to be organized in 2100 nt tandem arrays. RELP in the HSP60 genes revealed that at least three different types of HSP60 genes were encoded in the T cruzi genome. The predicted open reading frame measured exhibits about 50% identity to other HSP60 described. Expression of these HSP60 genes could not be induced by 2 hours heat shock at 37 degrees C. Post-transcriptional mechanisms may be responsible for HSP60 induction in T. cruzi.


Asunto(s)
Chaperonina 60/aislamiento & purificación , Genes Protozoarios/genética , Proteínas HSP70 de Choque Térmico/aislamiento & purificación , Proteínas Protozoarias/aislamiento & purificación , Trypanosoma cruzi/genética , Animales , Chaperonina 60/genética , Chaperonina 60/ultraestructura , Genoma de Protozoos , Proteínas HSP70 de Choque Térmico/genética , Proteínas HSP70 de Choque Térmico/ultraestructura , Proteínas Protozoarias/genética , Proteínas Protozoarias/ultraestructura
9.
Biol. Res ; 26(1/2): 313-4, 1993.
Artículo en Inglés | LILACS | ID: lil-228614

RESUMEN

To identify the members of the HSP70 and HSP60 families of Trypanosoma cruzi, we analysed 35S methionine epimastigote cells by two dimensional Western blot. At 29 degrees C, an HSP70 monoclonal antibody (anti-D. melanogaster) recognized eight isotypes. At least five of these were heat-induced. Polyclonal antibody against the 65 KDa antigen (anti-M. tuberculosis) recognized three isotypes with identical molecular weights, but different microliters. Only one isoform was heat induced. The cellular distribution of HSP70 and HSP60 was studied by immunoelectron microscopy. Anti-HSP70 reactive protein was localized in the cytoplasm, mitochondria and nucleus, while anti-HSP60 protein was found in the mitochondrion and in close association with the kinetoplast. To characterize the HSP60 gene and its proteins, we isolated a genomic T. cruzi clone encoding the HSP60 gene. T. cruzi HSP60 genes could be shown to be organized in 2100 nt tandem arrays. RELP in the HSP60 genes revealed that at least three different types of HSP60 genes were encoded in the T cruzi genome. The predicted open reading frame measured exhibits about 50 percent identity to other HSP60 described. Expression of these HSP60 genes could not be induced by 2 hours heat shock at 37 degrees C. Post-transcriptional mechanisms may be responsible for HSP60 induction in T. cruzi


Asunto(s)
Animales , Chaperonina 60/aislamiento & purificación , Genes Protozoarios/genética , Proteínas HSP70 de Choque Térmico/aislamiento & purificación , Proteínas Protozoarias/aislamiento & purificación , Trypanosoma cruzi/genética , Chaperonina 60/genética , Chaperonina 60/ultraestructura , Genoma de Protozoos , Proteínas HSP70 de Choque Térmico/genética , Proteínas HSP70 de Choque Térmico/ultraestructura , Proteínas Protozoarias/genética , Proteínas Protozoarias/ultraestructura
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