RESUMEN
Parasporin-4 (PS4) is an aerolysin-type ß-pore-forming toxin produced by Bacillus thuringiensis strain A1470. It exhibits specific cytotoxicity against human cancer cell lines; therefore, it is expected to be useful for the diagnosis and treatment of particular types of cancer cells. We examined the acute toxicity of PS4 on ICR mice. The LD50 value was 160 µg/kg by a subcutaneous route. Potassium, ammonium, magnesium ion, creatinine, and urea nitrogen decreased in urine by the injection of PS4. Simultaneously, creatinine and urea nitrogen in mice serum increased. These results imply that PS4 impairs kidney function in mice. PS4 is obtained from Pro-parasporin-4 (ProPS4) by processing, and ProPS4 is produced by recombinant Escherichia coli as the inclusion body. The inclusion body of ProPS4 can be solubilized in a weak acid solution and activated by pepsin, implying that it would be solubilized and activated in the stomach of mammals after oral administration. Thus, the influence of the oral administration of it by C57BL/6J mice was examined. Although ProPS4 was activated to PS4 in the mouse digestive tract, any serious health hazard was not observed and there was no significant difference in body weight change.
Asunto(s)
Proteínas Bacterianas/toxicidad , Endotoxinas/toxicidad , Compuestos de Amonio/orina , Animales , Nitrógeno de la Urea Sanguínea , Creatinina/sangre , Creatinina/orina , Dieta , Cuerpos de Inclusión , Riñón/efectos de los fármacos , Riñón/patología , Masculino , Ratones Endogámicos C57BL , Ratones Endogámicos ICR , Neoplasias/tratamiento farmacológico , Neoplasias/prevención & control , Potasio/orina , Proteínas Recombinantes , Sodio/orina , Urea/orinaRESUMEN
Parasporin is the cytocidal protein present in the parasporal inclusion of the non-insecticidal Bacillus thuringiensis strains, which has no hemolytic activity but has cytocidal activities, preferentially killing cancer cells. In this study, we characterized a cytocidal protein that belongs to this category, which was designated parasporin-5 (PS5). PS5 was purified from B. thuringiensis serovar tohokuensis strain A1100 based on its cytocidal activity against human leukemic T cells (MOLT-4). The 50% effective concentration (EC50) of PS5 to MOLT-4 cells was approximately 0.075 µg/mL. PS5 was expressed as a 33.8-kDa inactive precursor protein and exhibited cytocidal activity only when degraded by protease at the C-terminal into smaller molecules of 29.8 kDa. Although PS5 showed no significant homology with other known parasporins, a Position Specific Iterative-Basic Local Alignment Search Tool (PSI-BLAST) search revealed that the protein showed slight homology to, not only some B. thuringiensis Cry toxins, but also to aerolysin-type ß-pore-forming toxins (ß-PFTs). The recombinant PS5 protein could be obtained as an active protein only when it was expressed in a precursor followed by processing with proteinase K. The cytotoxic activities of the protein against various mammalian cell lines were evaluated. PS5 showed strong cytocidal activity to seven of 18 mammalian cell lines tested, and low to no cytotoxicity to the others.
Asunto(s)
Antineoplásicos/farmacología , Bacillus thuringiensis/metabolismo , Proteínas Bacterianas/farmacología , Endotoxinas/farmacología , Fragmentos de Péptidos/farmacología , Animales , Antineoplásicos/química , Antineoplásicos/aislamiento & purificación , Antineoplásicos/metabolismo , Proteínas Bacterianas/química , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Línea Celular , Línea Celular Tumoral , Supervivencia Celular/efectos de los fármacos , Chlorocebus aethiops , Cricetulus , Endopeptidasa K/metabolismo , Endotoxinas/química , Endotoxinas/genética , Endotoxinas/metabolismo , Humanos , Ratones , Peso Molecular , Concentración Osmolar , Fragmentos de Péptidos/química , Fragmentos de Péptidos/genética , Fragmentos de Péptidos/metabolismo , Isoformas de Proteínas/química , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Isoformas de Proteínas/farmacología , Proteolisis , Proteínas Recombinantes/efectos adversos , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Proteínas Recombinantes/farmacología , Homología de Secuencia de AminoácidoRESUMEN
Bacillus thuringiensis A1470 produces multiple proteins with similar molecular masses (~30 kDa) with cytotoxicity against human cell lines. One that was previously identified, parasporin-4, is a ß-pore-forming toxin. The N-terminal sequence of a second cytotoxic protein was identical to a partial sequence of parasporin-2 produced by B. thuringiensis A1547. PCR was performed on total plasmid DNA from A1470 by using primers for parasporin-2 to amplify a gene which was then cloned. The cloned gene differed from A1547 parasporin-2 by 8 bp and the predicted protein differed by four amino acids. The gene was expressed in Escherichia coli, and the cytotoxic activities of the recombinant protein against four human cell lines (MOLT-4, Jurkat, HeLa, and HepG2) were similar to those of A1547 parasporin-2. We then confirmed that the A1470 strain simultaneously produces parasporin-2 and parasporin-4.
Asunto(s)
Bacillus thuringiensis/metabolismo , Toxinas Bacterianas/metabolismo , Citotoxinas/metabolismo , Bacillus thuringiensis/genética , Toxinas Bacterianas/genética , Línea Celular , Clonación Molecular , Citotoxinas/genética , ADN Bacteriano/química , ADN Bacteriano/genética , Escherichia coli/genética , Expresión Génica , Humanos , Datos de Secuencia Molecular , Plásmidos , Proteínas Recombinantes/genética , Proteínas Recombinantes/aislamiento & purificación , Proteínas Recombinantes/toxicidad , Análisis de Secuencia de ADNRESUMEN
Two novel parasporin (PS) genes were cloned from Bacillus thuringiensis B0462 strain. One was 100 % identical even in nucleotide sequence level with that of parasporin-1Aa (PS1Aa1) from B. thuringiensis A1190 strain. The other (PS1Ac2) showed significant homology (99 % identity) to that of PS1Ac1 from B. thuringiensis 87-29 strain. The 15 kDa (S(113)-R(250)) and 60 kDa (I(251)-S(777)) fragments consisting of an active form of PS1Ac2 were expressed as His-tag fusion. Upon purification under denaturing condition and refolding, the recombinant polypeptides were applied to cancer cells to analyze their cytotoxicities. 3-(4,5-Dimethyl-2-thiazoyl)-2,5-diphenyl-2H-tetrazolium bromide assay revealed that either of 15 or 60 kDa polypeptide exhibited no cytotoxicity to HeLa cells, but they became cytotoxic upon mixed together. Our results suggested that PS1Ac2 was responsible for the cytotoxicity of B. thuringiensis B0462 strain, and that the formation of hetero-dimer of 15 and 60 kDa polypeptide was required for their cytotoxicity.
Asunto(s)
Bacillus thuringiensis/genética , Endotoxinas/genética , Bacillus thuringiensis/química , Endotoxinas/química , Endotoxinas/clasificación , Endotoxinas/aislamiento & purificación , Endotoxinas/toxicidad , Células HeLa , Humanos , Filogenia , Proteínas Recombinantes de Fusión/toxicidadRESUMEN
Parasporin-4 (PS4) is a cytotoxic protein produced by Bacillus thuringiensis strain A1470. It exhibits specific cytotoxicity against human cancer cell lines, CACO-2, Sawano, and MOLT-4 cells, in particular. When cells were administrated with PS4, cell swelling and nuclear shrinkage were induced, and, the ballooned cells burst within 24 h. PSI-BLAST search showed that the protein shared homology not only with B. thuringiensis Cry toxins but also with aerolysin-type ß-pore-forming toxins. Circular dichroism measurements suggested that PS4 was a ß-sheet-rich protein. PS4 aggregated into oligomers on the plasma membrane of PS4-susceptible CACO-2 cells, but not on that of PS4-resistant HeLa cells. Leakage of lactate dehydrogenase and influx of extracellular FITC-dextrans were observed only in susceptible cells. The activation of effectors caspase 3 and/or 7 was not observed in PS4-treated CACO-2 cells. It was shown that cytotoxicity of the PS4 against CACO-2 cells was exhibited when treated by cyclodextrin which induces cholesterol depletion. These results suggest that PS4 is a unique ß-pore-forming toxin with a cholesterol-independent activity.
Asunto(s)
Bacillus thuringiensis/metabolismo , Proteínas Bacterianas/farmacología , Endotoxinas/farmacología , Anticolesterolemiantes/farmacología , Proteínas Bacterianas/química , Proteínas Bacterianas/metabolismo , Células CACO-2 , Caspasa 3/metabolismo , Caspasa 7/metabolismo , Línea Celular Tumoral , Membrana Celular/efectos de los fármacos , Membrana Celular/metabolismo , Tamaño de la Célula/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Colesterol/metabolismo , Dicroismo Circular , Relación Dosis-Respuesta a Droga , Endotoxinas/química , Endotoxinas/metabolismo , Activación Enzimática/efectos de los fármacos , Citometría de Flujo , Células HeLa , Humanos , Células K562 , L-Lactato Deshidrogenasa/metabolismo , Lovastatina/farmacología , Microscopía de Contraste de Fase , Neoplasias/metabolismo , Neoplasias/patología , Unión Proteica , Multimerización de ProteínaRESUMEN
Bacillus thuringiensis strain M019, non-pathogenic to lepidopteran and dipteran insects, produces a parasporal inclusion that consists of three 84-kDa Cry proteins (CPs). CP78A and CP78B, which exhibit 83.5% amino acid identity, were new variants of the previously reported HeLa cell-killing protein (parasporin-1). CP84 was a novel CP showing low-level homology, of 21.9% (56.4% similarity), with the insecticidal Cry2 toxin. In vitro solubilization with dithiothreitol at pH 10.2 and limited hydrolysis with trypsin resulted in the removal of N-terminal portions of the CPs and their activation. The 70-kDa proteins (15- and 55-kDa fragments) from CP78A and CP78B and the 73-kDa protein (14- and 59-kDa fragments) from CP84 exhibited varying degrees of cytocidal activity preferentially toward human hepatocyte cancer HepG2 cells and uterus cervix cancer HeLa cells causing cell swelling or the formation of vacuoles in the cytoplasm. These toxins appeared to attack an identical target on human cells.
Asunto(s)
Antineoplásicos/farmacología , Bacillus thuringiensis/metabolismo , Proteínas Bacterianas/farmacología , Endotoxinas/farmacología , Proteínas Hemolisinas/farmacología , Hepatocitos/efectos de los fármacos , Neoplasias Hepáticas/patología , Neoplasias del Cuello Uterino/patología , Secuencia de Aminoácidos , Toxinas de Bacillus thuringiensis , Ditiotreitol/química , Femenino , Células HeLa , Células Hep G2 , Hepatocitos/patología , Humanos , Datos de Secuencia MolecularRESUMEN
The flagellated vegetative cells of the Bacillus thuringiensis strain K4 were agglutinated with the H3 reference antiserum and further, agglutinated with 3b and 3d monospecific factor sera but non-reactive for 3c and 3e factor sera. This creates a new serogroup with flagellar antigenic structure of 3a3b3d: B. thuringiensis serovar mogi. The strain K4 showed high activity against dipteran larvae, Anopheles sinensis and Culex pipienspallens while no lepidopteran toxicity. It produced ovoidal parasporal inclusions (crystals) whose SDS-PAGE protein profile consisted of several bands ranging from 75 to 30kDa. Through the protein identification by nano-LC-ESI-IT MS analysis, the putative peptides of Cry19Ba, Cry40ORF2, Cry27Aa and Cry20Aa were detected.
Asunto(s)
Anopheles/microbiología , Bacillus thuringiensis/fisiología , Proteínas Bacterianas , Culex/microbiología , Endotoxinas , Proteínas Hemolisinas , Insecticidas , Animales , Bacillus thuringiensis/clasificación , Bacillus thuringiensis/metabolismo , Toxinas de Bacillus thuringiensis , Control de Mosquitos , SerotipificaciónRESUMEN
Parasporin (PS) is a collection of genealogically heterogeneous Cry proteins synthesized in Bacillus thuringiensis. A prominent feature commonly associated with PS proteins is the strong cytocidal activity preferential for human cancer cells of various origins. The proteins exhibit cytocidal activities only when digested with proteases. Currently, this protein group is classified into four families: PS1, PS2, PS3 and PS4. Marked differences are evident in cytotoxicity spectra and activity levels between the four PS families. Neither hemolytic activity nor insect toxicity is associated with PS proteins. One of the most striking aspects in the events induced by PS1Aa1 is the early and rapid increase of the intracellular Ca2+ concentration, with no change in plasma membrane permeability. There is strong evidence that PS1Aa1 kills cancer cells through apoptosis. Unlike PS1Aa1, PS2Aa1 increases plasma membrane permeability of cancer cells. The initial step in cytocidal action of PS2Aa1 is the specific binding of this cytotoxin to a putative receptor located in the lipid rafts, followed by its oligomerization and pore formation in plasma membrane.
Asunto(s)
Antineoplásicos/farmacología , Bacillus thuringiensis/química , Endotoxinas/farmacología , Animales , Antineoplásicos/química , Endotoxinas/química , HumanosRESUMEN
Parasporin-2 is a protein toxin that is isolated from parasporal inclusions of the Gram-positive bacterium Bacillus thuringiensis. Although B. thuringiensis is generally known as a valuable source of insecticidal toxins, parasporin-2 is not insecticidal, but has a strong cytocidal activity in liver and colon cancer cells. The 37-kDa inactive nascent protein is proteolytically cleaved to the 30-kDa active form that loses both the N-terminal and the C-terminal segments. Accumulated cytological and biochemical observations on parasporin-2 imply that the protein is a pore-forming toxin. To confirm the hypothesis, we have determined the crystal structure of its active form at a resolution of 2.38 A. The protein is unusually elongated and mainly comprises long beta-strands aligned with its long axis. It is similar to aerolysin-type beta-pore-forming toxins, which strongly reinforce the pore-forming hypothesis. The molecule can be divided into three domains. Domain 1, comprising a small beta-sheet sandwiched by short alpha-helices, is probably the target-binding module. Two other domains are both beta-sandwiches and thought to be involved in oligomerization and pore formation. Domain 2 has a putative channel-forming beta-hairpin characteristic of aerolysin-type toxins. The surface of the protein has an extensive track of exposed side chains of serine and threonine residues. The track might orient the molecule on the cell membrane when domain 1 binds to the target until oligomerization and pore formation are initiated. The beta-hairpin has such a tight structure that it seems unlikely to reform as postulated in a recent model of pore formation developed for aerolysin-type toxins. A safety lock model is proposed as an inactivation mechanism by the N-terminal inhibitory segment.
Asunto(s)
Endotoxinas/química , Secuencia de Aminoácidos , Bacillus thuringiensis/química , Bacillus thuringiensis/metabolismo , Sitios de Unión , Cristalografía por Rayos X , Bases de Datos de Proteínas , Ensayos de Selección de Medicamentos Antitumorales , Modelos Moleculares , Datos de Secuencia Molecular , Conformación Proteica , Pliegue de Proteína , Serina/genética , Serina/metabolismo , Treonina/genética , Treonina/metabolismoRESUMEN
In 1901, a unique bacterium was isolated as a pathogen of the sotto disease of the silkmoth larvae, and later in 1915, the organism was described as Bacillus thuringiensis. Since the discovery, this bacterium has widely attracted attention of not only insect pathologists but many other scientists who are interested in strong and specific insecticidal activity associated with inclusion bodies of B. thuringiensis. This has led to the recent worldwide development of B. thuringiensis-based microbial insecticides and insect-resistant transgenic plants, as well as the epoch-making discovery of parasporin, a cancer cell-specific cytotoxin. In the review, we introduce a detection study of interaction between inclusion proteins of B. thuringiensis and brush border membrane of insects using surface plasmon resonance-based biosensor, and then identification and cloning of parasporin-4, a latest cancer cell-killing protein produced by B. thuringiensis A1470 strain. Inclusion bodies of the parasporin-4 produced by recombinant Escherichia coli were solubilized and activated with a new method and purified by an anion-exchange chromatography. At last the characterization of the recombinant parasporin-4 was shown.
Asunto(s)
Bacillus thuringiensis/clasificación , Bacillus thuringiensis/metabolismo , Membrana Celular/metabolismo , Endotoxinas/química , Endotoxinas/metabolismo , Insectos/metabolismo , Animales , Sitios de Unión , Unión Proteica , Especificidad de la EspecieRESUMEN
Four genes encoding parasporins, cytotoxins preferentially killing human cancer cells in vitro, were isolated from four Vietnamese strains of Bacillus thuringiensis. Nucleotide sequence analysis revealed that: (1) three genes fall into the two known classes, ps1Aa and ps1Ab, and (2) another one belongs to ps1Ac, a novel gene class established in this study. Upon proteolytic activation, parasporal protein of the organism with ps1Ac exhibited strong cytocidal activity against human cancer cells, HeLa and Hep G2, but not to non-cancer normal cells, UtSMC and HC.
Asunto(s)
Bacillus thuringiensis/genética , Endotoxinas/genética , Bacillus thuringiensis/aislamiento & purificación , Bacillus thuringiensis/patogenicidad , Endopeptidasa K/metabolismo , Humanos , Datos de Secuencia Molecular , Plásmidos , Reacción en Cadena de la Polimerasa , VietnamRESUMEN
The non-insecticidal Bacillus thuringiensis strain B0195 produces parasporin (PS) whose cytocidal activity is preferential for human cancer cells. This study identified two ps genes, ps1Aa3 and ps1Ab1, from the strain B0195. The former gene was 2,169-bp long, encoding an 81 kDa protein (PSLAa3) whose aminoacid sequence was 100% identical to that of the reference protein PS1Aa1. The latter gene was 2,178-bp long, encoding a novel protein of 82 kDa, PS1Ab1, whose sequence was 86.4% identical to that of PS1Aa1. The recombinant protein of PS1Ab1, synthesized in transformed B. thuringiensis cells, induced marked cytopathy in HeLa cells (human uterus cervix cancer cells) upon proteolytic activation. The cytopathy was characterized by cell-ballooning, followed by gradual cell-shrinking. Unlike HeLa cells, non-cancer UtSMC cells (human uterine smooth muscle-cells) were not susceptible to PS1Ab1.
Asunto(s)
Bacillus thuringiensis/genética , Endotoxinas/genética , Endotoxinas/farmacología , Secuencia de Bases , Clonación Molecular , Ensayos de Selección de Medicamentos Antitumorales , Células HeLa , Humanos , Datos de Secuencia Molecular , Proteínas Recombinantes/genética , Proteínas Recombinantes/farmacologíaRESUMEN
Parasporin, a Bacillus thuringiensis parasporal protein, is unique in having a strong cytocidal activity preferential for human cancer cells. In this study, we characterized parasporin activities associated with three novel geographical isolates of B. thuringiensis. Parasporal inclusion proteins of the three isolates were highly toxic to human uterus cervix cancer cells (HeLa), but not to non-cancer uterine smooth muscle cells (UtSMC). Inclusions of the isolates lacked insect toxicity and hemolytic activity against sheep erythrocytes. Ouchterlony immunodiffusion tests revealed that the proteins of the three isolates are immunologically closely related to parasporin-1 (Cry31A), but dissimilar to the three other existing parasporin groups. Our results provide evidence that the parasporin-1-producing organism is a common member in B. thuringiensis populations occurring in natural environments of Japan.
Asunto(s)
Bacillus thuringiensis/fisiología , Endotoxinas/biosíntesis , Pruebas de Aglutinación , Antineoplásicos/uso terapéutico , Antineoplásicos/toxicidad , Bacillus thuringiensis/ultraestructura , Proteínas Bacterianas/biosíntesis , Proteínas Bacterianas/uso terapéutico , Proteínas Bacterianas/toxicidad , Endotoxinas/uso terapéutico , Endotoxinas/toxicidad , Células HeLa/efectos de los fármacos , Humanos , Japón , Neoplasias/tratamiento farmacológicoRESUMEN
A novel gene encoding a leukemic cell-killing parasporal protein, designated parasporin-4, was cloned from an isolate of Bacillus thuringiensis serovar shandongiensis. The amino acid sequence of the parasporin-4, as deduced from the gene sequence, had low-level homologies of <30% with the established B. thuringiensis Cry proteins including the three known parasporins. When the gene was expressed in a recombinant of Escherichia coli BL21(DE3), the parasporin-4 formed intracellular inclusion bodies. Alkali-solubilized and proteinase K-activated inclusion protein exhibited strong cytotoxic activity against human leukemic T cells (MOLT-4) and weak for normal T cells, but no adverse effect on human uterus cervix cancer cells (HeLa).
Asunto(s)
Bacillus thuringiensis/genética , Muerte Celular/efectos de los fármacos , Endotoxinas/genética , Genes Bacterianos , Leucemia/patología , Secuencia de Bases , Línea Celular Tumoral , Cartilla de ADN , Relación Dosis-Respuesta a Droga , Ensayos de Selección de Medicamentos Antitumorales , Endotoxinas/farmacología , Humanos , Datos de Secuencia Molecular , Linfocitos T/efectos de los fármacosRESUMEN
Parasporin-2, a new crystal protein derived from noninsecticidal and nonhemolytic Bacillus thuringiensis, recognizes and kills human liver and colon cancer cells as well as some classes of human cultured cells. Here we report that a potent proteinase K-resistant parasporin-2 toxin shows specific binding to and a variety of cytocidal effects against human hepatocyte cancer cells. Cleavage of the N-terminal region of parasporin-2 was essential for the toxin activity, whereas C-terminal digestion was required for rapid cell injury. Protease-activated parasporin-2 induced remarkable morphological alterations, cell blebbing, cytoskeletal alterations, and mitochondrial and endoplasmic reticulum fragmentation. The plasma membrane permeability was increased immediately after the toxin treatment and most of the cytoplasmic proteins leaked from the cells, whereas mitochondrial and endoplasmic reticulum proteins remained in the intoxicated cells. Parasporin-2 selectively bound to cancer cells in slices of liver tumor tissues and susceptible human cultured cells and became localized in the plasma membrane until the cells were damaged. Thus, parasporin-2 acts as a cytolysin that permeabilizes the plasma membrane with target cell specificity and subsequently induces cell decay.
Asunto(s)
Antineoplásicos/farmacología , Bacillus thuringiensis/química , Toxinas Bacterianas/farmacología , Endotoxinas/metabolismo , Endotoxinas/farmacología , Neoplasias Hepáticas/metabolismo , Hígado/efectos de los fármacos , Animales , Antineoplásicos/metabolismo , Toxinas Bacterianas/genética , Toxinas Bacterianas/metabolismo , Membrana Celular/efectos de los fármacos , Forma de la Célula , Células Cultivadas , Citoesqueleto/efectos de los fármacos , Citoesqueleto/metabolismo , Endopeptidasa K/metabolismo , Endotoxinas/genética , Humanos , Hígado/citología , Hígado/metabolismo , Hígado/patología , Neoplasias Hepáticas/patología , Fragmentos de Péptidos/genética , Fragmentos de Péptidos/metabolismo , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismoRESUMEN
A total of 63 Bacillus thuringiensis isolates were recovered from urban soils of Hanoi, Vietnam. Of these, 34 were identified to 12 H serogroups. None of the isolates showed larvicidal activities against three lepidopterous insects. Three isolates belonging to the two serovars, colmeri (H21) and konkukian (H34), were highly toxic to larvae of the mosquito Aedes aegypti. Parasporal inclusion proteins of four isolates exhibited cytocidal activities against HeLa cells. Immunologically, proteins of four isolates were closely related to parasporin-1 (Cry31Aa), a parasporal protein that preferentially kills human cancer cells. Haemolytic activities were associated with parasporal proteins of the three mosquitocidal isolates but not with those of the four cancer-cell-killing isolates. PCR experiments and nucleotide sequence analysis revealed that the genes of four anti-cancer isolates are closely related to the gene parasporin-1 (cry31Aa) but are dissimilar to those of the three other existing parasporins. Our results suggest that the soil of northern Vietnam is a good reservoir of parasporin-producing B. thuringiensis.
Asunto(s)
Bacillus thuringiensis/metabolismo , Endotoxinas/biosíntesis , Animales , Antígenos Bacterianos/metabolismo , Bacillus thuringiensis/clasificación , Bacillus thuringiensis/genética , Proteínas Bacterianas/biosíntesis , Proteínas Bacterianas/genética , Proteínas Bacterianas/toxicidad , Supervivencia Celular/efectos de los fármacos , Endotoxinas/genética , Endotoxinas/toxicidad , Células HeLa , Hemólisis/efectos de los fármacos , Humanos , Cuerpos de Inclusión/química , Insectos/efectos de los fármacos , Microscopía de Contraste de Fase , Datos de Secuencia Molecular , Reacción en Cadena de la Polimerasa , Serotipificación , Ovinos , Microbiología del Suelo , VietnamAsunto(s)
Bacillus thuringiensis/metabolismo , Toxinas Bacterianas/química , Toxinas de Bacillus thuringiensis , Proteínas Bacterianas/química , Cristalización , Cristalografía por Rayos X , Endotoxinas/química , Proteínas Hemolisinas , Modelos Moleculares , Modelos Estadísticos , Datos de Secuencia Molecular , Conformación Proteica , Estructura Secundaria de Proteína , Espectrometría de Masa por Láser de Matriz Asistida de Ionización DesorciónRESUMEN
Gene transfer is an important tool to explore genomic, cell biologic, or gene therapeutic research. In this paper we report that several cationic amphiphiles have the potential to efficiently deliver DNA into CHO cells, which is one of the cell lines considered to be important for production of proteins including therapeutic proteins. We have found that O,O'-ditetradecanoyl-N-(trimethylammonio acetyl) diethanolamine chloride (14Dea2), among 29 types of cationic amphiphiles tested, shows a transfection efficiency of more than 40% in CHO cells. In addition, the results from a series of hydrocarbon chains of varying lengths bound to a connector have shown that an optimal chain length is important for the efficient delivery of DNA into cells. Moreover, flow cytometer analysis has shown that 14Dea2 transfection leads to high levels of expression of the reporter gene (green fluorescent protein) in individual cells. These findings have suggested that 14Dea2 is able to effectively deliver a number of plasmids into a cell nucleus. Thus, our system might be a powerful tool for high efficiency gene transfer and production of high levels of recombinant protein.
RESUMEN
A cytotoxic protein Cry45Aa of Bacillus thuringiensis expressed as inclusion bodies in Escherichia coli was solubilized in 10 mM HCl. Protein concentration of saturated solution of the recombinant Cry45Aa in 10 mM HCl was about 25 times higher than that in the buffer of previous method (in 50 mM sodium carbonate buffer, pH 10.5, containing 1 mM EDTA, and 10 mM dithiothreitol). The Cry45Aa solubilized in the acidic solution was activated by pepsin as an alternative to proteinase K in the previous method. Cytotoxic activity against CACO-2 cells of the pepsin-treated Cry45Aa was almost identical to the proteinase K-treated protein. The pepsin-treated Cry45Aa was purified by cation-exchange chromatography. The concentration of the purified protein was 539 microg/ml, which was 27-fold higher than that of the activated Cry45Aa by the previously method. The cytotoxic activity of the purified protein was stable in broad pH region (pH 2.0-11.0) for 3 days, and 97% cytotoxic activity remained after incubation at 30 degrees C for 360 min.