RESUMEN
In this study, we tried to decolorize synthetic dyes using small laccase (SLAC) from Streptomyces coelicolor, which is resistant to pH, temperature change, and traditional inhibitors for the actual industrial applications using spore surface display system. We inserted SLAC-His6 tag at the C-terminal of CotE anchoring motif. The proper surface expression of CotE-SLAC fusion protein on the surface of Bacillus subtilis spore was verified with flow cytometry using FITC labeled anti-His6 tag antibody. After 6 h of reaction, more than 90% of Indigo carmine was decomposed using recombinant SLAC displaying Bacillus spore, whereas less than 10% of Indigo carmine was decomposed with wild type spore. Over 70% of laccase activity was retained with recombinant SLAC displaying spore, which was heat-treated for 3 h at 90°C. For eight rounds of repeated decomposition of Indigo carmine, no significant decrease of enzymatic activity was observed. This showed the robust characteristics of spore display format for repeated and harsh condition reactions.
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Bacterial surface display system has been adopted in various biotechnological applications. In the case of Bacillus subtilis, most of the studies have been developed using spore based surface display system utilizing the inherent rigidity of spore against heat, alkali, and shear stress. But, spore harvest, purification and separation need additional cost and labor. To eliminate this procedure and to use the gram-positive nature of B. subtilis, YuaB, which is one of the major B. subtilis biofilm components and locates in the cell wall, based cell surface display system, is developed. P43 promoter driven overexpression of YuaB-His6 tag does not hamper bacterial cell growth and promoted biofilm formation of recombinant strain. Flow cytometry of recombinant strain and its protoplast using FITC-Anti His6 antibody, verified that YuaB locate in plasma membrane and protrude to the outside of cell wall, which means YuaB can be used as very efficient anchoring motif. Using surface expressed YuaB-His6 tag, removal of divalent metal ion, Cu2+ and Ni2+, was tried to test its possibility for the environmental application of developed system.
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Tyrosinases, copper-containing monooxygenases, are widely used enzymes for industrial, medical, and environmental applications. We report the first functional surface display of Bacillus megaterium tyrosinase on Bacillus subtilis spores using CotE as an anchor protein. Flow Cytometry was used to verify surface expression of tyrosinase on the purified spores. Moreover, tyrosinase activity of the displayed enzyme on B. subtilis spores was monitored in the presence of L-tyrosine (substrate) and CuSO4 (inducer). The stability of the spore-displayed tyrosinase was then evaluated after 15 days maintenance of the spores at room temperature, and no significant decrease in the enzyme activity was observed. In addition, the tyrosinase-expressing spores could be repeatedly used with 62% retained enzymatic activity after six times washing with Tris-HCl buffer. This genetically immobilized tyrosinase on the spores would make a new advance in industrial, medical, and environmental applications.
Asunto(s)
Bacillus megaterium/enzimología , Proteínas Bacterianas/genética , Técnicas de Visualización de Superficie Celular , Enzimas Inmovilizadas/genética , Monofenol Monooxigenasa/genética , Monofenol Monooxigenasa/metabolismo , Esporas Bacterianas/genética , Bacillus megaterium/genética , Bacillus subtilis/química , Bacillus subtilis/enzimología , Bacillus subtilis/genética , Estabilidad de Enzimas , Enzimas Inmovilizadas/metabolismo , Citometría de Flujo , Microbiología Industrial/métodos , Monofenol Monooxigenasa/química , Proteínas Recombinantes de Fusión/química , Proteínas Recombinantes de Fusión/metabolismo , Esporas Bacterianas/enzimología , Propiedades de Superficie , Tirosina/metabolismoRESUMEN
Surface display using spores of Bacillus subtilis is widely used to anchor antigens and enzymes of different sources. One open question is whether anchored proteins are able to form disulfide bonds. To answer this important question, we anchored the Escherichia coli alkaline phosphatase PhoA on the spore surface using two different surface proteins, CotB and CotZ. This enzyme needs two disulfide bonds to become active. Subsequently, we purified the spores and assayed for alkaline phosphatase activity. In both cases, we were able to recover enzymatic activity. Next, we asked whether formation of disulfide bonds occurs spontaneous or is catalyzed by thiol-disulfide oxidoreductases upon lysis of the cells. The experiment was repeated in a double-knockout mutant ΔbdbC and ΔbdbD. Since the disulfide bonds are also present on spores prepared from the double knockout, we conclude that oxidative environment after cell lysis is sufficient for disulfide formation of alkaline phosphatase.
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Bacillus subtilis/fisiología , Proteínas Bacterianas/metabolismo , Técnicas de Visualización de Superficie Celular , Disulfuros/metabolismo , Procesamiento Proteico-Postraduccional , Esporas Bacterianas/fisiología , Fosfatasa Alcalina/genética , Fosfatasa Alcalina/metabolismo , Bacillus subtilis/metabolismo , Escherichia coli/enzimología , Escherichia coli/genética , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Técnicas de Inactivación de Genes , Proteína Disulfuro Reductasa (Glutatión)/genética , Proteína Disulfuro Reductasa (Glutatión)/metabolismo , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Esporas Bacterianas/metabolismoRESUMEN
Sialic acids (SAs) are located on the terminal positions of glycan on a cell surface, which play important role in the spread and metastasis of cancer cells and infection of pathogen. For their detection and diagnosis, the finding of SA specific ligand is an essential prerequisite. Here, RNA aptamer for N-acetylneuraminic acid (Neu5Ac), a representative of SAs, with the high affinity of 1.35 nM and the selectivity was screened by in vitro selection method. The strong binding of the screened aptamer was enough to protect the hydrolysis of Neu5Ac by neuraminidase with the stoichiometry of 1:1 molar ratio. For the rapid detection of SAs, the RNA aptamer was further engineered to the aptazyme sensor by conjugating with a ribozyme following the characterization of selected aptamer by RNase footprinting assay. Without additional desialylation, modification, or/and purification processes, the aptazyme indicated high catalytic activities in the presence of Neu5Ac over 20 µM in several minutes. Also, we observed that the aptazyme sensor shows high sensitivities to Neu5Ac-conjugated sugars as well as Neu5Ac monomer, but not in non-Neu5Ac modified sugars. The aptamer for Neu5Ac can support valuable tools in a wide range of bioanalytical applications as well as biosensors.
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Aptámeros de Nucleótidos/metabolismo , Metabolismo de los Hidratos de Carbono , Ácidos Siálicos/análisis , Neuraminidasa/antagonistas & inhibidores , ARN Catalítico/metabolismoRESUMEN
For the functional bacterial surface display of active enzyme of multimeric form, which is generally impossible due to molecular assembly of the monomer subunit subsequent to the secretion of displayed target protein outside the cell, a new surface display system based on B. subtilis spore was developed. Using cotE and cotG of B. subtilis as anchoring motives, beta-galactosidase, which is active in tetrameric form, was functionally displayed on the surface of B. subtilis spore. The surface localization of beta-galactosidase was verified by Miller assay of purified spore, protease accessibility test of purified spore, and flow cytometric analysis of spore expressing beta-galactosidase. While B. subtilis spore wall integrity, examined by lysozyme and heat treatments, was affected by the incorporation of CotE-LacZ fusion protein, it was not affected by the incorporation of CotG-lacZ fusion. Heat stability of displayed protein was similar with that of free enzyme.
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Bacillus subtilis/crecimiento & desarrollo , Biopolímeros/química , Esporas Bacterianas , beta-Galactosidasa/química , Secuencia de Bases , Cartilla de ADN , Citometría de Flujo , Calor , Muramidasa/químicaRESUMEN
Recently Bacillus spp. has gained much attention as potential probiotics due to the production of resistant cells. So, this research is purposeful for evaluation of probiotic characteristics of Bacillus isolates from camel milk as a suitable source for growth and isolation of microorganisms that can be candidate to be used as probiotic. First, forty-eight colonies were screened by using morphological and biochemical analysis. Among the isolates, two of them were recognized as Bacillus subtilis CM1 and CM2 by partial 16SrRNA sequencing that, probiotic potentials of them were evaluated. Both of them, in the preliminary safety screening, were found negative for hemolysis and lecithinase activity. Also, in vitro characteristics such as acid, bile salts and artificial gastric juice resistant, cell surface hydrophobicity, auto-aggregation, antioxidant characteristics, and adherent capability to HT-29 cells were determined for them approximately in the range of other probiotic strains. Two strains were susceptible to various antibiotics and enterotoxigenic activities were not detected by PCR which means isolated Bacillus strains could be classified as safe. Altogether, results demonstrate that Bacillus CM1 and CM2 strains could have the potential of consideration as probiotics, however more extensive in vitro/vivo studies are needed.
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Bacillus subtilis , Bacillus , Animales , Camelus , Leche , Bacillus/genética , Antibacterianos/farmacologíaRESUMEN
Aerobic lactate production of which the final step is executed by lactate dehydrogenase (LDH) is one of the typical phenotypes in invasive tumor development. However, detailed mechanism of how LDH links to cancer cell invasiveness remains unclear. This study shows that suppressed LDHB expression plays a critical role in hepatoma cell invasiveness by inducing claudin-1 (Cln-1), a tight junction protein, via mitochondrial respiratory defects. First, we found that all the SNU human hepatoma cells with increased glycolytic lactate production have the defective mitochondrial respiratory activity and the Cln-1-mediated high invasive activity. Similar results were also obtained with human hepatocellular carcinoma tissues. Unexpectedly, the increased lactate production was due to LDH isozyme shifts to LDH5 by LDHB down-expression rather than LDHA induction, implying the importance of LDHB modulation. Second, LDHB knockdown did not only trigger Cln-1 induction at the transcriptional level, but also induced respiratory impairment. Interestingly, most respiratory inhibitors except KCN induced Cln-1 expression although complex I inhibition by rotenone was most effective on Cln-1 induction. Respiratory defect-mediated Cln-1 induction was further confirmed by knockdown of NDUFA9, one of complex I subunits. Finally, ectopic expression of LDHB attenuated the invasiveness of both SNU 354 and 449 cells whereas LDHB knockdown significantly augmented the invasiveness of Chang cells with Cln-1induction. The increased invasive activity by LDHB modulation was clearly reversed by knocking-down Cln-1. Taken together, our results suggest that LDHB suppression plays an important role in triggering or maintaining the mitochondrial defects and then contributes to cancer cell invasiveness by inducing Cln-1 protein.
Asunto(s)
Carcinoma Hepatocelular/patología , L-Lactato Deshidrogenasa/metabolismo , Neoplasias Hepáticas/patología , Proteínas de la Membrana/metabolismo , Mitocondrias/microbiología , Mitocondrias/patología , Invasividad Neoplásica , Línea Celular Tumoral , Respiración de la Célula/fisiología , Claudina-1 , Glucólisis/fisiología , Humanos , Isoenzimas/genética , Isoenzimas/metabolismo , L-Lactato Deshidrogenasa/genética , Ácido Láctico/metabolismo , ARN Interferente Pequeño/genética , ARN Interferente Pequeño/metabolismoRESUMEN
To improve the conventional bacterial surface display systems and to display a co-factor containing enzyme, ω-transaminase from Vibrio fluvialis, which needs pyridoxal phosphate (PLP) for efficient transamination, Bacillus subtilis spore display system with cotG, as an anchoring motif was used. Flow cytometry of the B. subtilis spore-expressing ω-transaminase proved its surface localization on the spore. The enzymatic activity of the spore expressing ω-transaminase was more than 30 times higher than that of the host spore. Protease treatment of the ω-transaminase displaying spores resulted in decreased transaminase activity, which is in keeping with the surface location of the fusion protein, CotG-ω-transaminase.
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Bacillus subtilis/genética , Proteínas Bacterianas/metabolismo , Ingeniería de Proteínas/métodos , Proteínas Recombinantes de Fusión/aislamiento & purificación , Esporas Bacterianas/genética , Transaminasas/aislamiento & purificación , Vibrio/enzimología , Bacillus subtilis/enzimología , Proteínas Bacterianas/genética , Bioensayo , Dimerización , Citometría de Flujo , Pliegue de Proteína , Fosfato de Piridoxal/metabolismo , Proteínas Recombinantes de Fusión/química , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo , Esporas Bacterianas/enzimología , Transaminasas/química , Transaminasas/genética , Transaminasas/metabolismo , Vibrio/químicaRESUMEN
Titanium iron (TiFe) alloy is a room-temperature hydrogen-storage material, and it absorbs hydrogen via a two-step process to form TiFeH and then TiFeH2. The effect of V addition in TiFe alloy was recently elucidated. The V substitution for Ti sublattice lowers P2/P1 ratio, where P1 and P2 are the equilibrium plateau pressure for TiFe/TiFeH and TiFeH/TiFeH2, respectively, and thus restricts the two-step hydrogenation within a narrow pressure range. The focus of the present investigation was to optimize the V content such that maximum usable storage capacity can be achieved for the target pressure range: 1 MPa for absorption and 0.1 MPa for desorption. The effect of V substitution at selective Ti or Fe sublattices was closely analyzed, and the alloy composition Ti46Fe47.5V6.5 displayed the best performance with ca. 1.5 wt.% of usable capacity within the target pressure range. At the same time, another issue in TiFe-based alloys, which is a difficulty in activation at room temperature, was solved by Ce addition. It was shown that 3 wt.% Ce dispersion in TiFe alloy imparted to it easy room-temperature (RT) activation properties.
RESUMEN
Polyhydroxyalkanoates (PHAs) are attractive new bioplastics for the replacement of plastics derived from fossil fuels. With their biodegradable properties, they have also recently been applied to the medical field. As poly(3-hydroxybutyrate) produced by wild-type Ralstonia eutropha has limitations with regard to its physical properties, it is advantageous to synthesize co- or terpolymers with medium-chain-length monomers. In this study, tung oil, which has antioxidant activity due to its 80% α-eleostearic acid content, was used as a carbon source and terpolymer P(53 mol% 3-hydroxybytyrate-co-2 mol% 3-hydroxyvalerate-co-45 mol% 3-hydroxyhexanoate) with a high proportion of 3-hydroxyhexanoate was produced in R. eutropha Re2133/pCB81. To avail the benefits of α-eleostearic acid in the tung oil-based medium, we performed partial harvesting of PHA by using a mild water wash to recover PHA and residual tung oil on the PHA film. This resulted in a film coated with residual tung oil, showing antioxidant activity. Here, we report the first application of tung oil as a substrate for PHA production, introducing a high proportion of hydroxyhexanoate monomer into the terpolymer. Additionally, the residual tung oil was used as an antioxidant coating, resulting in the production of bioactive PHA, expanding the applicability to the medical field.
RESUMEN
The targeting and anchoring of heterologous proteins and peptides to the outer surface of bacteriophages and cells is becoming increasingly important, and has been employed as a tool for fundamental and applied research in microbiology, molecular biology, vaccinology, and biotechnology. Less known are endospores or spores produced by some Gram-positive species. Spores of Bacillus subtilis are surrounded by a spore coat on their outside, and a few proteins have been identified being located on the outside layer and have been successfully used to immobilize antigens and some other proteins and enzymes. The major advantage of spores over the other published systems is their synthesis within the cytoplasm of the bacterial cell. Therefore, any heterologous protein to be anchored on the outside does not have to cross any membrane. Furthermore, spores are extremely resistant against high temperature, irradiation and many chemicals, and can be stored for many years at room temperature.
Asunto(s)
Bacillus subtilis/metabolismo , Proteínas Bacterianas/metabolismo , Bacillus subtilis/ultraestructura , Proteínas Recombinantes de Fusión/metabolismo , Esporas Bacterianas/metabolismo , Esporas Bacterianas/ultraestructuraRESUMEN
Transforming growth factor-ß (TGF-ß) acts as a key cytokine in epithelial-mesenchymal transition (EMT) and myofibroblast differentiation, which are important for normal tissue repair and fibrotic diseases. Ubiquitylation and proteasomal degradation of TGF-ß signaling proteins acts as a regulatory mechanism for the precise control of TGF-ß signaling. SMAD-specific ubiquitin E3 ligase (SMAD ubiquitination regulatory factor 2, SMURF2) controls TGF-ß signaling proteins including the TGF-ß receptor (TGFR) and SMAD2/3. Here, we report that tetratricopeptide repeat domain 3 (TTC3), a ubiquitin E3 ligase, positively regulates TGF-ß1-induced EMT and myofibroblast differentiation, through inducing ubiquitylation and proteasomal degradation of SMURF2. In human bronchial epithelial cells (BEAS-2B) and normal human lung fibroblasts, TTC3 knockdown suppressed TGF-ß1-induced EMT and myofibroblast differentiation, respectively. Similarly, when TTC3 expression was suppressed, the TGF-ß1-stimulated elevation of p-SMAD2, SMAD2, p-SMAD3, and SMAD3 were inhibited. In contrast, overexpression of TTC3 caused both EMT and myofibroblast differentiation in the absence of TGF-ß1 treatment. TGF-ß1 reduced SMURF2 levels and TTC3 overexpression led to a further decrease in SMURF2 levels, while TTC3 knockdown inhibited TGF-ß1-induced SMURF2 reduction. In cell and in vitro ubiquitylation assays demonstrated TTC3-mediated SMURF2 ubiquitylation, and coimmunoprecipitation assays established the binding between SMURF2 and TTC3. TGF-ß1-induced TTC3 expression was inhibited by the knockdown of SMAD2 and SMAD3. Finally, Ttc3 mRNA levels were significantly increased and Smurf2 protein levels were significantly decreased in the lungs of mice treated with bleomycin as compared with the lungs of control mice. Collectively, these data suggest that TTC3 may contribute to TGF-ß1-induced EMT and myofibroblast differentiation, potentially through SMURF2 ubiquitylation/proteasomal degradation and subsequent inhibition of SMURF2-mediated suppression of SMAD2 and SMAD3, which in turn induces TTC3 expression.
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Miofibroblastos/metabolismo , Factor de Crecimiento Transformador beta1/metabolismo , Ubiquitina-Proteína Ligasas/metabolismo , Animales , Bronquios/metabolismo , Bronquios/patología , Diferenciación Celular/fisiología , Células Epiteliales/metabolismo , Células Epiteliales/patología , Transición Epitelial-Mesenquimal , Células HEK293 , Humanos , Pulmón/metabolismo , Pulmón/patología , Masculino , Ratones , Miofibroblastos/patología , Transducción de Señal , Transfección , Factor de Crecimiento Transformador beta1/genética , Ubiquitina-Proteína Ligasas/genética , UbiquitinaciónRESUMEN
In this study, we expressed cotA laccase from Bacillus subtilis on the surface of B. subtilis spores for efficient decolorization of synthetic dyes. The cotE, cotG, and cotY genes were used as anchoring motifs for efficient spore surface display of cotA laccase. Moreover, a His6 tag was inserted at the C-terminal end of cotA for the immunological detection of the expressed fusion protein. Appropriate expression of the CotE-CotA (74 kDa), CotG-CotA (76 kDa), and CotY-CotA (73 kDa) fusion proteins was confirmed by western blot. We verified the surface expression of each fusion protein on B. subtilis spore by flow cytometry. The decoloration rates of Acid Green 25 (anthraquinone dye) for the recombinant DB104 (pSDJH-EA), DB104 (pSDJH-GA), DB104 (pSDJH-YA), and the control DB104 spores were 48.75%, 16.12%, 21.10%, and 9.96%, respectively. DB104 (pSDJH-EA) showed the highest decolorization of Acid Green 25 and was subsequently tested on other synthetic dyes with different structures. The decolorization rates of the DB104 (pSDJH-EA) spore for Acid Red 18 (azo dye) and indigo carmine (indigo dye) were 18.58% and 43.20%, respectively. The optimum temperature for the decolorization of Acid Green 25 by the DB104 (pSDJH-EA) spore was found to be 50°C. Upon treatment with known laccase inhibitors, including EDTA, SDS, and NaN3, the decolorization rate of Acid Green 25 by the DB104 (pSDJH-EA) spore decreased by 23%, 80%, and 36%, respectively.
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Antraquinonas/metabolismo , Bacillus subtilis/enzimología , Colorantes/metabolismo , Lacasa/metabolismo , Bacillus subtilis/genética , Bacillus subtilis/metabolismo , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Biodegradación Ambiental , Inhibidores Enzimáticos/química , Expresión Génica , Cinética , Lacasa/antagonistas & inhibidores , Lacasa/genética , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo , Esporas Bacterianas/enzimología , Esporas Bacterianas/genética , Esporas Bacterianas/metabolismo , TemperaturaRESUMEN
Purpose: Simple and cheap diagnostic kit development is one of the important aims of pharmaceutical developers and companies focused on public health improvement. The Bacillus subtilis spore surface-display technique is a genetic engineering method that is used to develop new-generation diagnostic kits applicable for the early detection of various types of diseases. In this study, we developed a novel simple, rapid, and inexpensive diagnostic paper-based kit to detect tyrosine in urine samples of humans and animals that is applicable for home or laboratory use. Methods: The B. subtilis spore-displayed tyrosinase system developed by genetic engineering methods was used to prepare a paper-based kit to detect tyrosine in urine samples of different groups of patients (i.e., patients with diabetes, diabetes with chronic kidney disease (CKD), and chronic kidney disease) for the detection of tyrosine during the acute disease phase. To confirm the sensitivity and specificity of the kit, tyrosine was also detected in urine samples using conventional liquid chromatography/mass spectroscopy. Results: Different concentrations of tyrosine (0.1-1 mM) were detected in urine samples based on visible changes of color from bright brownish-gray to dark brownish-gray within 1 hour. The kit could screen samples to distinguish the three groups of patients based on formation of a broad spectrum of colors reflecting the concentration of tyrosine. Conclusion: To the best of our knowledge, this is the first diagnostic kit with potential to rapidly diagnose various diseases related to the production of tyrosine in biological samples. This kit is not only widely applicable, including for personal use in the home, but is also appropriate as a part of standard screening tests and health protection programs in countries with limited resources.
RESUMEN
We present a thin membrane transducer (TMT) that can detect nucleic acid based biomolecular reactions including DNA hybridization and protein recognition by aptamers. Specific molecular interactions on an extremely thin and flexible membrane surface cause the deflection of the membrane due to surface stress change which can be measured by a compact capacitive circuit. A gold-coated thin PDMS membrane assembled with metal patterned glass substrate is used to realize the capacitive detection. It is demonstrated that perfect match and mismatch hybridizations can be sharply discriminated with a 16-mer DNA oligonucleotide immobilized on the gold-coated surface. While the mismatched sample caused little capacitance change, the perfectly matched sample caused a well-defined capacitance decrease vs. time due to an upward deformation of the membrane by a compressive surface stress. Additionally, the TMT demonstrated the single nucleotide polymorphism (SNP) capabilities which enabled a detection of mismatching base pairs in the middle of the sequence. It is intriguing that the increase of capacitance, therefore a downward deflection due to tensile stress, was observed with the internal double mismatch hybridization. We further present the detection of thrombin protein through ligand-receptor type recognition with 15-mer thrombin aptamer as a receptor. Key aspects of this detection such as the effect of concentration variation are investigated. This capacitive thin membrane transducer presents a completely new approach for detecting biomolecular reactions with high sensitivity and specificity without molecular labelling and optical measurement.
Asunto(s)
Aptámeros de Nucleótidos/análisis , Técnicas Biosensibles/métodos , ADN/análisis , Membranas Artificiales , Proteínas/análisis , Trombina/análisis , Aptámeros de Nucleótidos/química , Disparidad de Par Base/genética , Disparidad de Par Base/fisiología , Secuencia de Bases , Técnicas Biosensibles/instrumentación , ADN/química , Vidrio/química , Oro/química , Hibridación de Ácido Nucleico , Polimorfismo de Nucleótido Simple , Proteínas/química , Trombina/química , Factores de Tiempo , TransductoresRESUMEN
We first present a simple yet versatile strategy for the functionalization of polymer nanotubes in a controlled fashion. Carboxylic-acid-functionalized polypyrrole (CPPy) nanotubes were fabricated by using cylindrical micelle templates in a water-in-oil emulsion system, and the functional carboxyl groups were effectively incorporated into the polymer backbone during the polymerization by using pyrrole-3-carboxylic acid (P3CA) as a co-monomer without a sophisticated functionalization process. It was noteworthy that the chemical functionality of CPPy nanotubes was readily controlled in both qualitative and quantitative aspects. On the basis of the controlled functionality of CPPy nanotubes, a field-effect transistor (FET) sensor platform was constructed to detect specific biological entities by using a buffer solution as a liquid-ion gate. The CPPy nanotubes were covalently immobilized onto the microelectrode substrate to make a good electrical contact with the metal electrodes, and thrombin aptamers were bonded to the nanotube surface via covalent linkages as the molecular recognition element. The selective recognition ability of thrombin aptamers combined with the charge transport property of CPPy nanotubes enabled the direct and label-free electrical detection of thrombin proteins. Upon exposure to thrombin, the CPPy nanotube FET sensors showed a decrease in current flow, which was probably attributed to the dipole-dipole or dipole-charge interaction between thrombin proteins and the aptamer-conjugated polymer chains. Importantly, the sensor response was tuned by adjusting the chemical functionality of CPPy nanotubes. The efficacy of CPPy nanotube FET sensors was also demonstrated in human blood serum; this suggests that they may be used for practical diagnosis applications after further optimization.
Asunto(s)
Nanotubos/química , Polímeros/química , Proteínas/análisis , Proteínas/química , Pirroles/química , Calibración , Electroquímica , Microscopía Electrónica de Rastreo , Microscopía Electrónica de Transmisión , Estructura Molecular , Nanotubos/ultraestructuraRESUMEN
GroEL/S is a molecular chaperone system in Escherichia coli which not only assists the folding of intracellular proteins but also affects the cellular activity against the change of environmental condition. Here we show that the growth rate of E. coli DH5alpha can be improved at low temperature by expressing a GroEL/S variant achieved through irrational protein engineering approach. The GroELS variant (GroELS(var)) accelerating the growth of E. coli DH5alpha was screened through enrichment culture of the mutant libraries obtained by random mutagenesis. E. coli DH5alpha harboring the groELS(var) gene exhibited approximately 1.5-2 times higher growth rate compared to the strain with wild-type GroELS at 15-30 degrees C. At 10 degrees C, a temperature that the growth of E. coli DH5alpha almost stops, the GroELS(var) triggered the growth of E. coli DH5alpha. We identified that seven nucleotides of groELS gene and six amino acids of the GroELS were changed through the mutagenesis and screening. Site directed mutagenic analysis revealed that H360 in GroEL(var) is the most crucial residue determining the activity of GroELS(var) and more than one of the other residues in GroEL(var) may be additionally involved in the activity of GroELS(var). The improvement of growth rate induced by the GroELS(var) was observed only in the strain DH5alpha and not detected in other E. coli strains, such as BL21, BW25113, codon+, JM110, Top10, and XL1-blue.
Asunto(s)
Chaperonina 60/genética , Chaperonina 60/metabolismo , Escherichia coli/citología , Escherichia coli/fisiología , Mejoramiento Genético/métodos , Ingeniería de Proteínas/métodos , Proliferación Celular , Frío , Chaperonas Moleculares/genética , Chaperonas Moleculares/metabolismoRESUMEN
The fusion molecule (i.e. aptazyme) of aptamer and hammerhead ribozyme was developed as in situ sensor. Previously, the hammerhead ribozyme conjugated with aptamer through its stem II module showed a significant blank signal by self-cleavage. To reduce or remove its self-cleavage activity in the absence of target molecule, rational designs were attempted by reducing the binding affinity of the aptazyme to its RNA substrate, while maintaining the ribonuclease activity of the aptazyme. Interestingly, the bis-aptazymes which comprise the two aptamer-binding sites at both stem I and stem III of the hammerhead ribozyme showed very low blank signals, and their ratios of reaction rate constants, i.e. signal to noise ratios, were several tens to hundred times higher than those of the stem II-conjugated bis-aptazymes. The reduction in the blank signals seems to be caused by a higher dissociation constant between the main strand of the bis-aptazyme and its substrate arising from multi-point base-pairing of the bis-aptazymes. The bis-aptazymes for HCV replicase and helicase showed high selectivity against other proteins, and a linear relationship existed between their ribozyme activities and the target concentrations. In addition, a bis-aptazyme of dual functions was designed by inserting both aptamers for HCV replicase and helicase into the stem I and stem III of hammerhead ribozyme, respectively, and it also showed greater sensitivity and specificity for both proteins without blank signal.
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Aptámeros de Nucleótidos/química , Técnicas Biosensibles , Hepacivirus/enzimología , ARN Helicasas/análisis , ARN Catalítico/química , ARN Catalítico/metabolismo , ARN Polimerasa Dependiente del ARN/análisis , Aptámeros de Nucleótidos/metabolismo , ARN Helicasas/metabolismo , ARN Polimerasa Dependiente del ARN/metabolismo , Proteínas Virales/análisis , Proteínas Virales/metabolismoRESUMEN
To analyze a cotG-based Bacillus subtilis spore display system directly, GFP(uv) was expressed on the surface of Bacillus subtilis spores. When GFP(uv) was fused to the C-terminal of the cotG structural gene and expressed, the existence of a CotG-GFP(uv) fusion protein on the B. subtilis spore was confirmed by flow cytometry confocal microscopic analysis. When the cotG anchoring motif was deleted, no fluorescence emission was observed under flow cytometry and confocal microscopic analysis from the purified spore, confirming the essential role of CotG as an anchoring motif. This GFP(uv) displaying spore might be used for another signaling application triggered by intracellular or extracellular stimuli.