RESUMEN
Obtaining herbicide resistant plants is an important task in the genetic engineering of forest trees. Transgenic European aspen plants (Populus tremula L.) expressing the bar gene for phosphinothricin resistance have been produced using Agrobacterium tumefaciens-mediated transformation. Successful genetic transformation was confirmed by PCR analysis for thirteen lines derived from two elite genotypes. In 2014-2015, six lines were evaluated for resistance to herbicide treatment under semi-natural conditions. All selected transgenic lines were resistant to the herbicide Basta at doses equivalent to 10 l/ha (twofold normal field dosage) whereas the control plants died at 2.5 l/ha. Foliar NH4-N concentrations in transgenic plants did not change after treatment. Extremely low temperatures in the third ten-day period of October 2014 revealed differences in freeze tolerance between the lines obtained from Pt of f2 aspen genotypes. Stable expression of the bar gene after overwintering outdoors was confirmed by RT-PCR. On the basis of the tests, four transgenic aspen lines were selected. The bar gene could be used for retransformation of transgenic forest trees expressing valuable traits, such as increased productivity.
RESUMEN
We propose a new approach for the synthesis of biologically important nucleotides which includes a multi-enzymatic cascade conversion of D-pentoses into purine nucleotides. The approach exploits nucleic acid exchange enzymes from thermophilic microorganisms: ribokinase, phosphoribosylpyrophosphate synthetase, and adenine phosphoribosyltransferase. We cloned the ribokinase gene from Thermus sp. 2.9, as well as two different genes of phosphoribosylpyrophosphate synthetase (PRPP-synthetase) and the adenine phosphoribosyltransferase (APR-transferase) gene from Thermus thermophilus HB27 into the expression vectors, generated high-yield E. coli producer strains, developed methods for the purification of the enzymes, and investigated enzyme substrate specificity. The enzymes were used for the conversion of D-pentoses into 5-phosphates that were further converted into 5-phospho-α-D-pentofuranose 1-pyrophosphates by means of ribokinase and PRPP-synthetases. Target nucleotides were obtained through the condensation of the pyrophosphates with adenine and its derivatives in a reaction catalyzed by APR-transferase. 2-Chloro- and 2-fluoroadenosine monophosphates were synthesized from D-ribose and appropriate heterobases in one pot using a system of thermophilic enzymes in the presence of ATP, ribokinase, PRPP-synthetase, and APR-transferase.
Asunto(s)
Antivirales/farmacología , Virus de la Influenza A/efectos de los fármacos , Infecciones por Orthomyxoviridae/tratamiento farmacológico , Oseltamivir/farmacología , Ribavirina/farmacología , Replicación Viral/efectos de los fármacos , Amantadina/farmacología , Animales , Antivirales/administración & dosificación , Antivirales/uso terapéutico , Perros , Combinación de Medicamentos , Farmacorresistencia Viral , Virus de la Influenza A/fisiología , Células de Riñón Canino Madin Darby , Ratones , Oseltamivir/administración & dosificación , Oseltamivir/uso terapéutico , Ribavirina/administración & dosificación , Ribavirina/uso terapéuticoRESUMEN
The potentialities and restrictions of chemoenzymatic approach to the synthesis of new structural analogues of antiviral drug Ribavirin (1-beta-D-ribofuranosyl-1,2,4-triazole-3-carboxamide) have been determined. Syntheses of various amides of 1H-1,2,4-triazole-3-carboxylic acid and its 5-substituted analogues, prospective substrates of purine nucleoside phosphorylase (PNP), have been reported. The comparative effectiveness of the methods for obtaining amides aforementioned and also the methods for introducing functional groups to the C5 position of the heterocyclic system has been studied. New Ribavirin analogues bearing various substituents in the carboxamide group have been synthesized. The biotechnological method for the preparation of 1-beta-D-ribofuranosyl- 1,2,4-triazole-3-carbonitrile used as the intermediate in the synthesis of Viramidine, a contemporary Ribavirin analogue, has been developed.
Asunto(s)
Nucleósidos/síntesis química , Ribavirina/síntesis química , Triazoles/síntesis química , Antivirales/síntesis química , Humanos , Nucleósidos/química , Purina-Nucleósido Fosforilasa , Ribavirina/análogos & derivados , Ribavirina/química , Ribonucleósidos/síntesis química , Especificidad por Sustrato , Triazoles/químicaRESUMEN
Improved biotechnological method of receiving the antiviral drug ribavirin by the reaction of transglycosilation by addition of catalytic amounts of sodium arsenate in the reaction mixture. Such approach allows to hydrolyze the amount of the excess natural nucleoside donor--ribose and, as a consequence, to simplify the composition of the reaction mixture and the process of separation of ribavirin. The effect of ribavirin and ozeltamivir carboxylate and their combination on the reproduction of the virus of the influenza A in cell culture and in experiments on laboratory animals (mouse Balb/C). The greatest anti-influenza effect is observed when using a combination of drugs, as compared to each of them taken separately.
Asunto(s)
Virus de la Influenza A/efectos de los fármacos , Gripe Humana/tratamiento farmacológico , Ribavirina/administración & dosificación , Replicación Viral/efectos de los fármacos , Animales , Arseniatos/síntesis química , Arseniatos/química , Perros , Combinación de Medicamentos , Humanos , Gripe Humana/virología , Células de Riñón Canino Madin Darby , Ratones , Oseltamivir/administración & dosificación , Ribavirina/análogos & derivados , Ribavirina/síntesis químicaRESUMEN
Pigment epithelium-derived factor (PEDF), a 50 kDa secreted glycoprotein, is among the most potent endogenous inhibitors of angiogenesis. PEDF-derived fragment (44-77) possesses antiangiogenic properties of the full-sized protein and is a potential drug candidate for the treatment of ocular neovascular diseases. In this study we propose an efficient scalable biotechnological method for the production of PEDF (44-77) as part of a fusion protein with SspDnaB intein. The fusion protein was obtained in bacterial E. coli cells in the form of inclusion bodies, solubilized and subjected to autocatalytic cleavage with the release of PEDF (44-77) (yield, 77%). The target peptide was separated from the intein using tangential ultrafiltration. The final purification of PEDF (44-77) was performed by reversed-phase HPLC. The yield of the target peptide (purity, 99%) was 65 mg per 1 liter of culture. Antiangiogenic activity of the obtained peptide was studied in vitro using murine endothelial cells SVEC-4-10. PEDF (44-77) suppressed proliferation of endothelial cells by 53% and inhibited endothelial cell tube formation at the concentration of 1 nM. The ability of the recombinant PEDF (44-77) to block initial stages of angiogenesis was demonstrated using the model of rabbit corneal neovascularization.
Asunto(s)
Inhibidores de la Angiogénesis/biosíntesis , Inhibidores de la Angiogénesis/farmacología , Neovascularización de la Córnea/prevención & control , Proteínas del Ojo/biosíntesis , Proteínas del Ojo/farmacología , Factores de Crecimiento Nervioso/biosíntesis , Factores de Crecimiento Nervioso/farmacología , Serpinas/biosíntesis , Serpinas/farmacología , Inhibidores de la Angiogénesis/genética , Animales , Línea Celular , Proliferación Celular/efectos de los fármacos , Neovascularización de la Córnea/tratamiento farmacológico , Neovascularización de la Córnea/genética , Neovascularización de la Córnea/metabolismo , Neovascularización de la Córnea/patología , Modelos Animales de Enfermedad , Células Endoteliales/metabolismo , Células Endoteliales/patología , Escherichia coli/genética , Escherichia coli/metabolismo , Proteínas del Ojo/genética , Humanos , Ratones , Factores de Crecimiento Nervioso/genética , Conejos , Proteínas Recombinantes/biosíntesis , Proteínas Recombinantes/genética , Proteínas Recombinantes/farmacología , Serpinas/genéticaRESUMEN
Hirudin-1 is a highly selective inhibitor of thrombin secreted by salivary glands of the medicinal leech Hirudo medicinalis. This direct anticoagulant is used for the treatment and prevention of disorders in blood coagulation system. Apart from the existing recombinant analog of hirudin-1 (63-desulfatohirudin-1, desirudin) its modified analogs possessing higher activity and stability are of medical value. In this study artificial genes of hirudin and two its analogs (hirudin-1, [Leu1, Thr2]-hirudin-1 and [Leu1, Thr2]-hirudin-1/3) were synthesized and cloned in an expression vector pTWIN1 in frame with the gene of mini-intein SspDnaB from Synechocystis sp. Producing strains of the corresponding fusion proteins were constructed using E. coli strain ER2566. Biotechnological schemes for the production of 63-desulfatohirudin-1 and its analogs were developed. The scheme includes the following stages: isolation of the fusion protein after the desintegration of the cell biomass, refolding of the target peptide within the fusion protein, pH-inducible cleavage of the fusion protein, and chromatographic purification of the target product. Antithrombotic activity of the obtained peptides was determined by a standard amidolytic assay. The developed methods for the production of 63-desulfatohirudin-1, [Leu1, Thr2]-desulfatohirudin-1 [Leu1, Thr2]-desulfatohirudin-1/3 allowed to obtain these peptides with high yields (14, 25 and 24 mg per liter of cell culture respectively) and high activity (13423, 33333 and 19802 ATU/mg respectively).
Asunto(s)
Biotecnología/métodos , Hirudinas , Hirudo medicinalis , Animales , Escherichia coli/genética , Escherichia coli/metabolismo , Hirudinas/biosíntesis , Hirudinas/química , Hirudinas/genética , Hirudinas/aislamiento & purificación , Proteínas Recombinantes/biosíntesis , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/aislamiento & purificaciónRESUMEN
A thymidine kinase UL23 gene (EC 2.7.1.145) from an acyclovir-sensitive strain L2 of herpes simplex virus type 1 was cloned and expressed in E. coli. Enzyme was purified by chromatography to a homogeneous state controlled by PAG electrophoresis. The Michaelis constants for the reactions with thymidine and an acyclovir were determined. It was found that enzyme phosphorilate some modified nucleosides such as d2T, d4T, d2C, 3TC, FLT, BVDU, GCV. A comparison of the purified enzyme properties and properties ofthymidine kinase of other strains of herpes simplex virus, previously published was carried out. It is shown that enzyme is inhibited by acyclovir H-phosphonate.
Asunto(s)
Aciclovir/farmacología , Simplexvirus/enzimología , Timidina Quinasa/metabolismo , Secuencia de Aminoácidos , Antivirales/metabolismo , Antivirales/farmacología , Clonación Molecular , Escherichia coli/genética , Expresión Génica , Humanos , Datos de Secuencia Molecular , Mutación , Análisis de Secuencia de ADN , Timidina Quinasa/antagonistas & inhibidores , Timidina Quinasa/química , Timidina Quinasa/aislamiento & purificaciónRESUMEN
A method for isolation of interferon beta1b (Serl7) from inclusion bodies, comprising the steps of solution and reduction of protein from the inclusion bodies, refolding, chromatography on DEAE-Sepharose, chromatography on SP-Sepharose, concentrating, desalting and addition of stabilizers. The solution of reduced protein was diluted with pH 8.0 buffer of 50 mM Tris-HCl, 25 microM CuCl2 and 0.5% Twin 20 for refolding. We used gradient of pH (from 9.3 upto 11.3) for elution of interferon-beta from cation-exchange column. We concentrated of eluate and then desalted on the Sephadex G-50 column with 1 mM NaOH. Then the protein solution was neutralized with mannitol and Na-phosphate. Obtained preparation of interferon-beta was pure by gel-electrophoresis and by HPLC analysis, and had practically indentical level of antiproliferative activity with well-known preparation of Betaferone. Thus we show the possibility of isolation and obtaining of pure and active interferone-beta by ion-exchange chromatography in the presence of non-ion detergent Twin 20. We believe this method for interferon betalb preparation is perspective for scaling and using in the develop of industrial technology for production of this preparation.
Asunto(s)
Interferón beta/aislamiento & purificación , Proteínas Recombinantes/aislamiento & purificación , Cromatografía por Intercambio Iónico/métodos , Escherichia coli/genética , Expresión Génica , Humanos , Cuerpos de Inclusión/química , Interferon beta-1b , Interferón beta/genética , Proteínas Recombinantes/genéticaRESUMEN
Thymosin beta4 (43 aa) is a highly conserved acidic peptide which regulates actin polymerization in mammalian cells by sequestering globular actin. Thymosin beta4 is undergoing clinical trials as a drug for the treatment of venous stasis ulcers, corneal wounds and injuries, as well as acute myocardial infarction. Currently, thymosin beta4 is produced with solid-phase chemical synthesis. Biotechnological synthesis of this peptide presents difficulties because N-terminal amino acid residue of thymosin beta4 is acetylated. In this study we propose a method for producing the recombinant precursor of thymosin beta4 and its subsequent targeted chemical acetylation. Desacetylthymosin beta4 was synthesized as a part of a hybrid protein with thioredoxin and a specific TEV (tobacco etch virus) protease cleavage site. The following scheme was developed for the purification of desacetylthymosin beta4: (i) the biosynthesis of a soluble hybrid protein (HP) in Escherichia coli; (ii) isolation of the HP by ion exchange chromatography; (iii) cleavage of the HP with TEVprotease; (iv) purification of desacetylthymosin beta4 by ultra-filtration. N-terminal acetylation of desacetylthymosin beta4 was performed with acetic anhydride under acidic conditions (pH 3). The reaction yield was 55%. Thymosin beta4 was then purified by reverse-phase high performance liquid chromatography. The proposed synthetic approach to recombinant thymosin beta4 is suitable for scale-up and can provide for the medical use of highly purified preparation with a yield of 20 mg from 1 L of culture.
Asunto(s)
Enfermedades de la Córnea/tratamiento farmacológico , Infarto del Miocardio/tratamiento farmacológico , Timosina/metabolismo , Timosina/uso terapéutico , Úlcera Varicosa/tratamiento farmacológico , Anhídridos Acéticos/química , Acetilación , Biotecnología/métodos , Cromatografía Líquida de Alta Presión , Humanos , Concentración de Iones de Hidrógeno , Proteínas Recombinantes de Fusión/genética , Tiorredoxinas/genética , Tiorredoxinas/metabolismo , Timosina/síntesis química , Timosina/genéticaRESUMEN
A real-time PCR procedure is proposed for assaying E. coli residual DNA in the pharmaceutical substance of human recombinant insulin. For the quantitative analysis of the DNA content, an amplification of fragments of the bla gene plasmid DNA and E. coli genomic DNA of the 16S RNA gene were used. The contents of plasmid and genomic DNA were detected both in intermediates at various stages of the insulin purification process and in the finished product.
Asunto(s)
ADN Bacteriano/análisis , Insulina/química , Escherichia coli/genética , Humanos , Reacción en Cadena de la Polimerasa , Ingeniería de Proteínas , Sensibilidad y Especificidad , beta-Lactamasas/genéticaRESUMEN
The ability of water preparations of different composition to affect the generation of superoxide radicals has been compared. The superoxygenating reaction of adrenaline autoxidation with some modifications was used as a model system, which makes it possible to reveal the pro/antioxidant properties of materials being tested. It was shown that samples of water from sources having different specific electroconductivity and, accordingly, ionic composition differ in the ability to affect reactions proceeding with the participation of ROS. The parameter measured, the pro/antioxidant activity of water, is a new informative indicator, and the approach proposed enables one to perform a comparative estimation of the quality of water and aqueous solutions.
Asunto(s)
Antioxidantes/química , Agua Dulce/química , Oxidantes/química , Conductividad Eléctrica , Epinefrina/química , Concentración de Iones de Hidrógeno , Soluciones , Superóxidos/químicaRESUMEN
The cointegration rate into the aspen and birch genomes of foreign genes from a binary vector and a disarmed Ti plasmid pCBE21 carried by the same Agrobacterium tumefaciens strain was studied. The cotransformation rate for the genes within the Ti plasmid varied from 30 to 100%; while the transformation rate for the gene from T(L) region was twofold higher as compared with the T(R) region. On the average, the gene transfer from all three T-DNAs was recorded in 10.9% of the transgenic lines. For the vector pBI121, the cotransformation rates for the genes from both regions of pCBE21 T-DNA were higher as compared with the vector pGS. In addition, a concurrent transfer of the genes from the Ti plasmid T(L) and T(R) regions was recorded only after the transformation with the vector pBI121. These results can be used for constructing woody plants containing several genes.
Asunto(s)
Agrobacterium tumefaciens/genética , Betula/genética , ADN Bacteriano/genética , Plantas Modificadas Genéticamente/genética , Populus/genética , Genes Bacterianos , Vectores Genéticos , Glutamato-Amoníaco Ligasa/genética , Plásmidos , Transformación GenéticaRESUMEN
This review focuses on new trends in nucleoside biotechnology, which have emerged during the last decade. Continuously growing interest in the study of this class of compounds is fueled by a number of factors: ( i ) a growing need for large-scale production of natural 2 ' -deoxy- ß -D-ribonucleosides as well as their analogs with modifications in the carbohydrate and base fragments, which can then be used for the synthesis and study of oligonucleotides, including short-interfering RNA (siRNA), microRNA (miRNA), etc.; ( ii ) a necessity for the development of efficient practical technologies for the production of biologically important analogs of natural nucleosides, including a number of anticancer and antiviral drugs; ( iii ) a need for further study of known and novel enzymatic transformations and their use as tools for the efficient synthesis of new nucloside analogs and derivates with biomedical potential. This article will review all of these aspects and also include a brief retrospect of this field of research.
RESUMEN
Conditions were found at the analytical level for the solubilization of a recombinant insulin precursor from inclusion bodies in different buffer systems at a wide pH range in the presence of different reducing (dithiothreitol, dithioerythritol) and chaotropic agents (urea, guanidine hydrochloride) and the subsequent renaturation with the use of redox pairs (cysteine-cystine, oxidized glutathione-reduced glutathione, and others). The scaling of the method for the production of the active substance of genetically engineered human insulin has been performed.
Asunto(s)
Industria Farmacéutica/métodos , Escherichia coli/metabolismo , Proinsulina/aislamiento & purificación , Proteínas Recombinantes/aislamiento & purificación , Electroforesis en Gel de Poliacrilamida , Humanos , Cuerpos de Inclusión/metabolismo , Proinsulina/biosíntesis , Desnaturalización Proteica , Pliegue de Proteína , Renaturación de Proteína , Proteínas Recombinantes/biosíntesis , Solubilidad , Compuestos de Sulfhidrilo/metabolismoRESUMEN
A preparation of nanocomplexes containing recombinant proteins (interferons alpha2b and beta1b, insulin, and human granulocyte colony stimulating factor) and natural polysialic acid (PSA) has been described. The incorporation of protein into the complex changes its electrophoretic mobility. Atomic force microscopy reveals the average size of 23-kD insulin complexes with PSA of 10-20 nm and demonstrates that more than 60% of glycopolymer molecules carry a single protein molecule. Experiments with cultured cells show that cytokines bound to polysialic acid retain their ability to regulate cell proliferation. Insulin bound to PSA has a prolonged hypoglycemic effect in vivo.
Asunto(s)
Factor Estimulante de Colonias de Granulocitos/química , Insulina/química , Interferón-alfa/química , Interferón beta/química , Nanoestructuras , Ácidos Siálicos/química , Animales , Antineoplásicos/química , Antineoplásicos/farmacología , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Electroforesis en Gel de Poliacrilamida , Factor Estimulante de Colonias de Granulocitos/farmacología , Humanos , Hipoglucemiantes/química , Hipoglucemiantes/farmacología , Insulina/farmacología , Interferón alfa-2 , Interferon beta-1b , Interferón-alfa/farmacología , Interferón beta/farmacología , Ratones , Microscopía de Fuerza Atómica , Conejos , Proteínas Recombinantes/química , Proteínas Recombinantes/farmacología , Ácidos Siálicos/farmacologíaRESUMEN
Methods of selective and nonselective covalent immobilization of genetically engineered proteins on molecules of natural polysialic acid are described by the example of human insulin. Such modification increases insulin lifetime in vivo.
Asunto(s)
Insulina/química , Ácidos Siálicos/química , Humanos , Proteínas Recombinantes/químicaRESUMEN
The ability of Bacillus licheniformis strain VKM B-511 to grow and synthesize biosurfactants under both aerobic and anaerobic conditions has been demonstrated. Yields of biosurfactants, emulsion indices and surface tension were considerably higher in culture liquor and preparations derived from cultures grown anaerobically at a C/N ratio of 1 : 24, pH 7.0, and temperature of 30 degrees C. Biosurfactant production by B. lichenformis also depended on concentrations of NaCl and Na2S in the medium and on water characteristics, reaching 4.58 g/l for bacteria grown anaerobically on a medium containing anolyte fraction of water.
Asunto(s)
Bacillus/crecimiento & desarrollo , Bacillus/metabolismo , Medios de Cultivo/química , Tensoactivos/metabolismo , Aerobiosis/fisiología , Anaerobiosis/fisiología , Calor , Concentración de Iones de HidrógenoRESUMEN
Bacteriophage T5 deoxynucleoside monophosphate kinase (dNMP kinase, EC 2.7.4.13) is shown to catalyze the phosphorylation of both d(2)CMP and ribonucleotides AMP, GMP, and CMP, but does not phosphorylate UMP. For natural acceptors of the phosphoryl group, K(m) and k(cat) were found. The applicability of T5 dNMP kinase as a universal enzyme capable of the phosphorylation of labelled r/dNMP was shown for the synthesis of [alpha-(32)P]rNTP and [alpha-(32)P]dNTP.
Asunto(s)
Nucleótidos/química , Fosfotransferasas (Aceptor del Grupo Fosfato)/química , Siphoviridae/enzimología , Proteínas Virales/química , Marcaje Isotópico/métodos , Isótopos de Fósforo/química , Especificidad por SustratoRESUMEN
Natural and modified purine nucleosides have been synthesized using the recombinant thermostable enzymes purine nucleoside phosphorylase II (EC 2.4.2.1) and pyrimidine nucleoside phosphorylase (EC 2.4.2.2) from Geobacillus stearothermophilus B-2194. The enzymes were produced in recombinant E. coli strains and covalently immobilized on aminopropylsilochrom AP-CPG-170 after heating the cell lysates and the removal of coagulated thermolabile proteins. The resulting preparations of thermostable nucleoside phosphorylases retained a high activity after 20 reuses in nucleoside transglycosylation reactions at 70-75 degrees C with a yield of the target products as high as 96%. Owing to the high catalytic activity, thermal stability, the ease of application, and the possibility of repeated use, the immobilized preparations of thermostable nucleoside phosphorylases are suitable for the production of pharmacologically important natural and modified nucleosides.