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1.
Bull Exp Biol Med ; 167(3): 375-379, 2019 Jul.
Artículo en Inglés | MEDLINE | ID: mdl-31346881

RESUMEN

In the process of optimization of heterologous expression of thermostable chemotaxis proteins CheW and CheY as industrially useful polypeptides, their direct influence on the cell growth kinetics and morphology of Escherichia coli was observed. CheW and CheY of bacteria of the genus Thermotoga, being expressed in recombinant form in E. coli cells, are involved in the corresponding signal pathways of the mesophilic microorganisms. The effects of such involvement in the metabolism of "host" cells are extremely diverse: from rapid aging of the culture to elongation of the stationary growth phase. We also discuss the mechanisms of the influence of the heterologous chemotaxis proteins on cells, their positive and negative effects, as well as potential applications in industry and biomedicine.


Asunto(s)
Bacterias/genética , Proteínas Bacterianas/biosíntesis , Escherichia coli/metabolismo , Proteínas Quimiotácticas Aceptoras de Metilo/biosíntesis , Proteínas Bacterianas/genética , Reactores Biológicos/microbiología , Quimiotaxis/genética , Escherichia coli/genética , Proteínas de Escherichia coli , Expresión Génica/genética , Proteínas Quimiotácticas Aceptoras de Metilo/genética , Thermotoga
2.
Biomed Khim ; 65(1): 41-50, 2019 Jan.
Artículo en Ruso | MEDLINE | ID: mdl-30816096

RESUMEN

Using genetic engineering methods the expression vectors structures have been designed to produce recombinant proteins TnaCheY and Tna CheY-mut, the homologues of the chemotaxis protein CheY from the hyperthermophilic organism Thermotoga naphthophila in Escherichia coli BL21(DE3) cells. The cultivation conditions of transformed strains were optimized. The influence of episomal expression of the heterologous chemotaxis protein CheY on growth kinetics parameters of the culture of mesophilic bacteria E. coli was studied. The optimal purification flowchart of the obtained proteins using thermolysis is proposed. Using the E. coli BL21(DE3) laboratory strain as an example, the possibility of employment the episomal expression of such proteins to control the cultivation and production time of pharmaceutically and industrially valuable metabolites due to the impact on some stages of the bacterial chemotaxis is experimentally proved.


Asunto(s)
Proteínas Bacterianas/genética , Quimiotaxis , Bacilos Gramnegativos Anaerobios Rectos, Curvos y Espirales/genética , Proteínas Quimiotácticas Aceptoras de Metilo/genética , Escherichia coli , Proteínas Mutantes/genética , Proteínas Recombinantes
3.
Bull Exp Biol Med ; 165(4): 461-464, 2018 Aug.
Artículo en Inglés | MEDLINE | ID: mdl-30121908

RESUMEN

Mutant homologues of small chemotactic and DNA-binding proteins from thermophilic bacteria Thermotoga petrophila RKU-1 and Thermotoga naphthophila were obtained. These proteins can be expressed in the recombinant form in E. coli cells. A wide range of properties and parameters that are important for isolation of these proteins were revealed: stability in a wide temperature and pH range, high level of expression, solubility, and the possibility of using simple purification schemes with low number of successive steps. The positive effect of proteins on in vitro fibroblasts growth was demonstrated. The described properties of the target proteins indicate the possibility of their use in different biotechnology industries as an inexpensive source of L-amino acids.


Asunto(s)
Aminoácidos/química , Aminoácidos/metabolismo , Medios de Cultivo/química , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Fibroblastos/metabolismo , Humanos , Concentración de Iones de Hidrógeno , Temperatura
4.
Bull Exp Biol Med ; 164(4): 478-482, 2018 Mar.
Artículo en Inglés | MEDLINE | ID: mdl-29504103

RESUMEN

The effect of apoptotic endonuclease EndoG on alternative splicing of mRNA of human telomerase catalytic subunit hTERT (human telomerase reverse transcriptase) and telomerase activity in normal human lymphocytes were studied. Human CD4+, CD8+, B, and NK cells were transfected with a plasmid pEndoG-GFP containing EndoG gene or control plasmid pGFP. The levels of mRNA of EndoG or hTERT splicing variants were analyzed by real-time PCR. Protein content was assessed by Western blotting. Telomerase activity was measured by the telomere repeats amplification protocol. EndoG overexpression reduced the expression of active full-length hTERT and increased the expression of inactive splice variant. Shifted balance of hTERT splice variants in cells led to a significant decrease in telomerase activity within 72 h after transfection.


Asunto(s)
Empalme Alternativo , Linfocitos B/inmunología , Linfocitos T CD4-Positivos/inmunología , Linfocitos T CD8-positivos/inmunología , Endonucleasas/genética , Células Asesinas Naturales/inmunología , Telomerasa/genética , Apoptosis/genética , Apoptosis/inmunología , Linfocitos B/citología , Linfocitos T CD4-Positivos/citología , Linfocitos T CD8-positivos/citología , Endonucleasas/inmunología , Regulación Enzimológica de la Expresión Génica , Genes Reporteros , Proteínas Fluorescentes Verdes/genética , Proteínas Fluorescentes Verdes/metabolismo , Humanos , Inmunofenotipificación , Células Asesinas Naturales/citología , Transducción de Señal , Telomerasa/inmunología
5.
Biomed Khim ; 64(1): 53-60, 2018 Jan.
Artículo en Ruso | MEDLINE | ID: mdl-29460835

RESUMEN

In the work a recombinant chemotaxis protein CheW from Thermotoga petrophila RKU-1 (TpeCheW) and its mutant homolog (TpeCheW-mut) were created. It was shown that, despite the low homology with CheW prototypes from intestinal bacteria, these proteins didn't cause metabolic overload and were well expressed by cells of E. coli laboratory strains. We have discovered a broad spectrum of industrial valuable properties of the TpeCheW-mut protein such as stability in a wide range of temperatures and pH, high expression level, solubility and possibility of the application of a simple low-stage purification methodology with the use of preliminary heat treatment. Possible directions of the scientific and industrial application of this protein were claimed.


Asunto(s)
Bacilos Gramnegativos Anaerobios Rectos, Curvos y Espirales , Proteínas Bacterianas , Escherichia coli , Proteínas de Escherichia coli , Proteínas Recombinantes
6.
Biomed Khim ; 63(4): 296-305, 2017 Jul.
Artículo en Ruso | MEDLINE | ID: mdl-28862599

RESUMEN

Activity of telomerase catalytic subunit hTERT (human Telomerase Reverse Transcriptase) can be regulated by alternative splicing of its mRNA. At present time exact mechanism of hTERT splicing is not fully understood. Apoptotic endonuclease EndoG is known to participate this process. EndoG expression is induced by DNA damages. The aim of this work was to investigate the ability of DNA-damaging agents with different mechanism of action to induce EndoG expression and inhibit telomerase activity due to the activation of hTERT alternative splicing in normal activated human CD4+ and CD8+ T-lymphocytes. All investigated DNA-damaging agents were able to induce EndoG expression. Cisplatin, a therapeutic compound, producing DNA cross-links induced the highest level of DNA damages and EndoG expression. Incubation of CD4+ and CD8+ T-cells with cisplatin caused the changes in proportion of hTERT splice variants and inhibition of telomerase activity.


Asunto(s)
Empalme Alternativo , Linfocitos T CD4-Positivos/enzimología , Linfocitos T CD8-positivos/enzimología , Daño del ADN , Endodesoxirribonucleasas/metabolismo , Telomerasa/genética , Células Cultivadas , Cisplatino , Humanos
7.
Biochemistry (Mosc) ; 82(8): 894-905, 2017 Aug.
Artículo en Inglés | MEDLINE | ID: mdl-28941457

RESUMEN

The activity of telomerase catalytic subunit hTERT (human telomerase reverse transcriptase) can be regulated by alternative splicing of its mRNA. The mechanism of hTERT splicing is not understood in detail. Apoptotic endonuclease EndoG is known to participate in this process. In the present work, the intracellular colocalization and mRNA levels of EndoG and hTERT splice-variants in normal and apoptotic cancer cells were studied. We found that the development of apoptosis increased the expression of EndoG and changed the ratio of hTERT splice-variants, which decreased the telomerase activity in the cells. The development of apoptosis was accompanied by changes in the amount of mRNA and in the localization of EndoG and hTERT splice-variants in the cytoplasm, nuclei, and mitochondria of the cells. The suppression of EndoG expression using RNA interference prevented induction of the α+ß- splice-variant of hTERT and inhibition of the telomerase activity. A high degree of the intracellular colocalization of EndoG and hTERT was shown. The changes in the expression and localization of EndoG corresponded with changes in the amount and localization of hTERT splice-variants. These data confirm the participation of EndoG in the alternative splicing of mRNA of the telomerase catalytic subunit and in regulation of the telomerase activity.


Asunto(s)
Endonucleasas/metabolismo , Telomerasa/metabolismo , Empalme Alternativo , Apoptosis , Células CACO-2 , Dominio Catalítico , Endonucleasas/antagonistas & inhibidores , Endonucleasas/genética , Humanos , Transporte de Proteínas , Interferencia de ARN , ARN Mensajero/metabolismo , ARN Interferente Pequeño/metabolismo , Telomerasa/química , Telomerasa/genética
8.
Biomed Khim ; 63(2): 124-131, 2017 Mar.
Artículo en Ruso | MEDLINE | ID: mdl-28414283

RESUMEN

The construction of proteins and peptides with desired properties, including resistance to high temperatures, as well as optimization of their amino acid composition, is an important and complex task, which attracts much attention in various branches of the basic sciences, and also in biomedicine and biotechnology. This raises the question: what method is more relevant for the at the pilot stage of research in order to estimate the influence of the planned amino acid substitutions on the thermostability of the resultant protein construct? In this brief review we have classified existing basic practical and theoretical approaches used in studies and predicting the thermal stability of native and recombinant polypeptides. Particular attention has been paid to the predictive potential of statistical methods for studying the thermodynamic parameters of the primary protein structure and prospects of their use.


Asunto(s)
Algoritmos , Aminoácidos/química , Ingeniería de Proteínas , Proteínas/química , Secuencia de Aminoácidos , Sustitución de Aminoácidos , Animales , Archaea/química , Bacterias/química , Calor , Humanos , Estabilidad Proteica , Estructura Secundaria de Proteína , Estructura Terciaria de Proteína , Proteínas/ultraestructura , Termodinámica
9.
Biomed Khim ; 63(1): 13-26, 2017 Jan.
Artículo en Ruso | MEDLINE | ID: mdl-28251947

RESUMEN

Alternative splicing of telomerase catalytic subunit hTERT pre-mRNA (human Telomerase Reverse Transcriptase) regulates telomerase activity. Increased expression of non-active splice variant hTERT results in inhibition of telomerase. Apoptotic endonuclease EndoG is known to participate in hTERT alternative splicing. Expression of EndoG can be induced in response to DNA damages. The aim of this study was to determine the ability of a DNA-damaging compound, cisplatin, to induce EndoG and its influence on alternative splicing of hTERT and telomerase activity in human CD4+ Т lymphocytes. Overexpression of EndoG in CD4+ T cells downregulated the expression of active full-length hTERT variant and upregulated its non-active spliced variant. Reduction of full-length hTERT caused downregulation of telomerase activity, shortening of telomeres length during cell divisions, converting cells to the replicative senescence state, activation of apoptosis and finally cell death. Few cells survived and underwent malignant transformation. Transformed cells have increased telomerase activity and proliferative potential compare to initial CD4+ T cells. These cells have phenotype of T lymphoblastic leukemic cells and are able to form tumors and cause death in experimental mice.


Asunto(s)
Antineoplásicos/toxicidad , Linfocitos T CD4-Positivos/efectos de los fármacos , Transformación Celular Neoplásica/genética , Cisplatino/toxicidad , Endodesoxirribonucleasas/genética , Telomerasa/genética , Empalme Alternativo/efectos de los fármacos , Animales , Apoptosis , Linfocitos T CD4-Positivos/inmunología , Linfocitos T CD4-Positivos/patología , Linfocitos T CD4-Positivos/trasplante , Proliferación Celular/efectos de los fármacos , Transformación Celular Neoplásica/inducido químicamente , Transformación Celular Neoplásica/inmunología , Transformación Celular Neoplásica/patología , Endodesoxirribonucleasas/metabolismo , Humanos , Linfoma/genética , Linfoma/inmunología , Linfoma/mortalidad , Linfoma/patología , Masculino , Ratones , Ratones Endogámicos BALB C , Ratones Desnudos , Cultivo Primario de Células , Análisis de Supervivencia , Telomerasa/antagonistas & inhibidores , Telomerasa/metabolismo , Telómero/química , Telómero/efectos de los fármacos , Acortamiento del Telómero/efectos de los fármacos
10.
Biomed Khim ; 63(1): 62-74, 2017 Jan.
Artículo en Ruso | MEDLINE | ID: mdl-28251953

RESUMEN

The active and stable mutant forms of short chain cytoplasmic L-asparaginase type I of Rhodospirillum rubrum (RrA): RrA+N17, D60K, F61L, RrA+N17, A64V, E67K, RrA+N17, E149R, V150P, RrAE149R, V150P and RrAE149R, V150P, F151T were obtained by the method of site-directed mutagenesis. It is established that variants RrA-N17, E149R, V150P, F151T and RrАE149R, V150P are capable to reduce an expression hTERT subunit of telomerase and, hence, activity of telomeres in Jurkat cells, but not in cellular lysates. During too time, L-asparaginases of Escherichia coli, Erwinia carotovora and Wolinella succinogenes, mutant forms RrА+N17, D60K, F61L and RrА+N17, A64V, E67K do not suppress of telomerase activity. The assumption of existence in structure RrA of areas (amino acids residues in the position 146-164, 1-17, 60-67) which are responsible for suppression of telomerase activity is made. The received results show that antineoplastic activity of some variants RrA is connected both with reduction of concentration of free L-asparagine, and with expression suppression of hTERT telomerase subunit, that opens new prospects for antineoplastic therapy.


Asunto(s)
Antineoplásicos/farmacología , Asparaginasa/farmacología , Proteínas Bacterianas/farmacología , Mutación Puntual , Rhodospirillum rubrum/enzimología , Telomerasa/antagonistas & inhibidores , Telómero/efectos de los fármacos , Secuencia de Aminoácidos , Antineoplásicos/química , Antineoplásicos/metabolismo , Asparaginasa/química , Asparaginasa/genética , Asparaginasa/metabolismo , Proteínas Bacterianas/química , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Línea Celular Tumoral , Supervivencia Celular/efectos de los fármacos , Escherichia coli/química , Escherichia coli/enzimología , Escherichia coli/genética , Expresión Génica , Células HL-60 , Humanos , Células Jurkat , Modelos Moleculares , Mutagénesis Sitio-Dirigida , Pectobacterium carotovorum/química , Pectobacterium carotovorum/enzimología , Pectobacterium carotovorum/genética , Plásmidos/química , Plásmidos/metabolismo , Estructura Secundaria de Proteína , Rhodospirillum rubrum/química , Rhodospirillum rubrum/genética , Especificidad de la Especie , Relación Estructura-Actividad , Telomerasa/genética , Telomerasa/metabolismo , Telómero/química , Wolinella/química , Wolinella/enzimología , Wolinella/genética
11.
Biochemistry (Mosc) ; 82(1): 24-37, 2017 Jan.
Artículo en Inglés | MEDLINE | ID: mdl-28320284

RESUMEN

Telomerase activity is regulated by an alternative splicing of mRNA of the telomerase catalytic subunit hTERT (human telomerase reverse transcriptase). Increased expression of the inactive spliced hTERT results in inhibition of telomerase activity. Little is known about the mechanism of hTERT mRNA alternative splicing. This study was aimed at determining the effect of an apoptotic endonuclease G (EndoG) on alternative splicing of hTERT and telomerase activity in CD4+ human T lymphocytes. Overexpression of EndoG in CD4+ T cells downregulated the expression of the active full-length hTERT variant and upregulated the inactive alternatively spliced variant. Reduction of full-length hTERT levels caused downregulation of the telomerase activity, critical telomere shortening during cell division that converted cells into the replicative senescence state, activation of apoptosis, and finally cell death. Some cells survive and undergo a malignant transformation. Transformed cells feature increased telomerase activity and proliferative potential compared to the original CD4+ T cells. These cells have phenotype of T lymphoblastic leukemia cells and can form tumors and cause death in experimental mice.


Asunto(s)
Empalme Alternativo , Linfocitos T CD4-Positivos/enzimología , Transformación Celular Neoplásica/metabolismo , Endodesoxirribonucleasas/biosíntesis , Regulación Enzimológica de la Expresión Génica , Regulación Neoplásica de la Expresión Génica , Proteínas de Neoplasias/biosíntesis , Leucemia-Linfoma Linfoblástico de Células T Precursoras/enzimología , Telomerasa/biosíntesis , Homeostasis del Telómero , Animales , Linfocitos T CD4-Positivos/patología , Transformación Celular Neoplásica/genética , Transformación Celular Neoplásica/patología , Endodesoxirribonucleasas/genética , Femenino , Xenoinjertos , Humanos , Masculino , Ratones , Proteínas de Neoplasias/genética , Trasplante de Neoplasias , Leucemia-Linfoma Linfoblástico de Células T Precursoras/genética , Leucemia-Linfoma Linfoblástico de Células T Precursoras/patología , Telomerasa/genética
12.
Prikl Biokhim Mikrobiol ; 53(2): 164-72, 2017.
Artículo en Ruso | MEDLINE | ID: mdl-29508977

RESUMEN

N-hydroxysuccinimide ester of monomethoxy polyethylene glycol hemisuccinate was synthesized. It acylated amino groups in a molecule of recombinant L-asparaginase from Erwinia carotovora. A method of L-asparaginase modification by the obtained activated polyethylene glycol derivative was developed. The best results were produced by modification of the enzyme with a 25-fold excess of reagent relative to the enzyme tetramer. The modified L-asparaginase was isolated from the reaction mixture by gel filtration on Sepharose CL-6B. The purified bioconjugate did not contain PEG unbound to the protein, demonstrated high catalytic activity, and exhibited antiproliferative action on cell cultures.


Asunto(s)
Antineoplásicos Fitogénicos/química , Asparaginasa/química , Proteínas Bacterianas/química , Pectobacterium carotovorum/química , Polietilenglicoles/química , Antineoplásicos Fitogénicos/biosíntesis , Antineoplásicos Fitogénicos/farmacología , Asparaginasa/biosíntesis , Asparaginasa/genética , Asparaginasa/farmacología , Proteínas Bacterianas/biosíntesis , Proteínas Bacterianas/genética , Proteínas Bacterianas/farmacología , Supervivencia Celular/efectos de los fármacos , Cromatografía en Gel , Clonación Molecular , Reactivos de Enlaces Cruzados/química , Escherichia coli/genética , Escherichia coli/metabolismo , Expresión Génica , Células HL-60 , Humanos , Células Jurkat , Células K562 , Pectobacterium carotovorum/enzimología , Polietilenglicoles/farmacología , Ingeniería de Proteínas , Proteínas Recombinantes/biosíntesis , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/farmacología , Succinimidas/química
13.
Vopr Pitan ; 86(3): 19-31, 2017.
Artículo en Ruso | MEDLINE | ID: mdl-30645859

RESUMEN

Proteins and polypeptides play a key role in the life of a human body. Scientific and practical interest to the natural proteinaceous substances could be explained by the diversity of their functions in metabolic processes. Biologically active substances of the protein origin have a rich history of applications in different sectors of the economy. In this case, the close relationship is observed between food industry, biomedicine and fodder production, because efficient conversion of feed protein in productive agricultural animals provides, as a result, the required level of metabolism in a human, as the main consumer of final products derived from these animals. Obviously, for normal growth, development and resistance to infectious agents, both people and farm animals need a constant consumption of L-amino acids in certain proportions and in available for absorption form. This review considers the bioactive polypeptides used in nutrition and food industry, main trends and practical approaches to generating protein products with the desired characteristics.

14.
Biomed Khim ; 62(5): 544-554, 2016 Jul.
Artículo en Ruso | MEDLINE | ID: mdl-27797329

RESUMEN

Human telomerase catalytic subunit hTERT is subjected to alternative splicing results in loss of its function and leads to decrease of telomerase activity. However, very little is known about the mechanism of hTERT pre-mRNA alternative splicing. Apoptotic endonuclease EndoG is known to participate this process. The aim of this study was to determine the role of EndoG in regulation of hTERT alternative splicing. Increased expression of b-deletion splice variant was determined during EndoG over-expression in CaCo-2 cell line, after EndoG treatment of cell cytoplasm and nuclei and after nuclei incubation with EndoG digested cell RNA. hTERT alternative splicing was induced by 47-mer RNA oligonucleotide in naked nuclei and in cells after transfection. Identified long non-coding RNA, that is the precursor of 47-mer RNA oligonucleotide. Its size is 1754 nucleotides. Based on the results the following mechanism was proposed. hTERT pre-mRNA is transcribed from coding DNA strand while long non-coding RNA is transcribed from template strand of hTERT gene. EndoG digests long non-coding RNA and produces 47-mer RNA oligonucleotide complementary to hTERT pre-mRNA exon 8 and intron 8 junction place. Interaction of 47-mer RNA oligonucleotide and hTERT pre-mRNA causes alternative splicing.


Asunto(s)
Empalme Alternativo/fisiología , Endodesoxirribonucleasas/metabolismo , Exones , ARN no Traducido/biosíntesis , Telomerasa/biosíntesis , Células CACO-2 , Endodesoxirribonucleasas/genética , Humanos , ARN no Traducido/genética , Telomerasa/genética
15.
Biomed Khim ; 62(3): 239-50, 2016 Mar.
Artículo en Ruso | MEDLINE | ID: mdl-27420614

RESUMEN

Telomerase activity is known to be regulated by alternative splicing of its catalytic subunit hTERT (human Telomerase Reverse Transcriptase) mRNA. Induction of non-active spliced hTERT leads to inhibition of telomerase activity. However, very little is known about the mechanism of hTERT mRNA alternative splicing. The aim of this study was to determine the role of apoptotic endonuclease EndoG in alternative splicing of hTERT and telomerase activity. Strong correlation was found between expression of EndoG and hTERT splice-variants in 12 colon cancer cell lines. Overexpression of EndoG in СаСо-2 cells downregulated the expression of active full-length hTERT variant and upregulated non-active spliced variant. Reduction of full-length hTERT caused downregulation of telomerase activity, dramatically shortening of telomeres length during cell divisions, converting cells to the replicative senescence state, activation of apoptosis and finally cell death. These data indicated the participation of EndoG in alternative splicing of mRNA of telomerase catalytic subunit, regulation of telomerase activity and cell fate.


Asunto(s)
Empalme Alternativo , Apoptosis , Endodesoxirribonucleasas/metabolismo , Telomerasa/genética , Células CACO-2 , Endodesoxirribonucleasas/genética , Células HCT116 , Células HT29 , Humanos , Telomerasa/metabolismo
16.
Biomed Khim ; 61(3): 312-24, 2015.
Artículo en Ruso | MEDLINE | ID: mdl-26215408

RESUMEN

For more than 40 years L-asparaginases are used in combined therapy of acute lymphoblastic leukemia in children and the range of tumors sensitive to these enzymes constantly extends. This review summarizes results of studies aimed at creation of new systems for heterological expression of bacterial L-asparaginases as Erwinia carotovora (EwA), Helicobacter pylori (HpA), Yersinia pseudotuberculosis (YpA) and Rhodospirillum rubrum (RrA); special attention is paid to isolation of purified enzymes and their crystallization, modification by chitosan/polyethylene, physicochemical, kinetic and structural properties characterization, and the study of the cytotoxic or anti-proliferative activity of new recombinant L-asparaginases on cell cultures in vitro. The resultant recombinant L-asparaginases (EwA, YpA, HpA и RrA) exhibit reasonable cytotoxic action on the human leukemia cells comparable to the pharmacologically available L-asparaginase EcA and represent practical interest in respect to creation, on their basis, new effective antineoplastic remedies. Further prospects of researches on bacterial L-asparaginases are associated with development of analogs of Rhodospirillum rubrum L-asparaginase (RrA) by means of directed changes of the protein structure using genetic engineering, development of chito-PEGylation for receiving L-asparaginase preparations with improved pharmacokinetic characteristics.


Asunto(s)
Antineoplásicos/farmacología , Asparaginasa/química , Asparaginasa/farmacología , Proteínas Bacterianas/farmacología , Secuencia de Aminoácidos , Antineoplásicos/química , Asparaginasa/genética , Proteínas Bacterianas/química , Proteínas Bacterianas/genética , Línea Celular Tumoral/efectos de los fármacos , Helicobacter pylori/enzimología , Humanos , Leucemia/tratamiento farmacológico , Leucemia/patología , Datos de Secuencia Molecular , Pectobacterium carotovorum/enzimología , Ingeniería de Proteínas/métodos , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/aislamiento & purificación , Proteínas Recombinantes/farmacología , Rhodospirillum rubrum/enzimología , Yersinia pseudotuberculosis/enzimología
17.
Mol Biotechnol ; 57(3): 251-64, 2015 Mar.
Artículo en Inglés | MEDLINE | ID: mdl-25370827

RESUMEN

Site-directed mutagenesis of Rhodospirillum rubrum L-asparaginase (RrA) was performed in order to identify sites of the protein molecule important for its therapeutic and physico-chemical properties. Ten multipoint mutant genes were obtained, and five recombinant RrA variants were expressed in E. coli BL21(DE3) cells and isolated as functionally active highly purified proteins. Protein purification was performed using Q-Sepharose and DEAE-Toyopearl chromatography. Overall yield of the active enzymes was 70-80 %, their specific activity at pH 7.4 and 37 °C varied of 140-210 U/mg. L-Glutaminase activity did not exceed 0.01 % of L-asparaginase activity. All RrA mutants showed maximum enzyme activity at pH 9.3-9.5 and 53-58 °C. Km and Vmax values for L-asparagine were evaluated for all mutants. Mutations G86P, D88H, M90K (RrAH), G121L, D123A (RrАI) caused the loss of enzyme activity and confirmed the importance of these sites in the implementation of catalytic functions. Removal of four residues from C-terminal area of the enzyme (RrAK) resulted in the enzyme instability. Mutations D60K, F61L(RrАD), and R118H, G120R(RrАJ) led to the improvement of kinetic parameters and enzyme stabilization. Substitutions E149R, V150P (RrАB) improved antineoplastic and cytotoxic activity of the RrA. A64V, E67K substitutions, especially in combination with E149R, V150P (RrАE), considerably destabilized recombinant enzyme.


Asunto(s)
Asparaginasa/química , Asparaginasa/genética , Proteínas Bacterianas/química , Proteínas Bacterianas/genética , Mutagénesis Sitio-Dirigida/métodos , Rhodospirillum rubrum/enzimología , Antineoplásicos/metabolismo , Antineoplásicos/farmacología , Asparaginasa/biosíntesis , Asparaginasa/farmacología , Proteínas Bacterianas/biosíntesis , Proteínas Bacterianas/farmacología , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Clonación Molecular , Estabilidad de Enzimas , Escherichia coli/genética , Humanos , Modelos Moleculares , Leucemia-Linfoma Linfoblástico de Células T Precursoras/tratamiento farmacológico , Rhodospirillum rubrum/genética
18.
Artículo en Ruso | MEDLINE | ID: mdl-25816523

RESUMEN

AIM: Evaluate immune response in mice against various L-asparaginases and determine their cross-immunogenicity. MATERIALS AND METHODS: The studies were carried out in C57Bl(6j) line mice. Immunogenicity of L-asparaginases was studied: Escherichia coli type II (recombinant) (Medak, Germany) (EcA); Erwinia carotovora type II (ErA); Yersinia pseudotuberculosis type II (YpA); Rhodospirillum rubrum type I (RrA); Wollinella succinogenes type II (WsA). Immune response against the administered antigens was determined in EIA. RESULTS: Y. pseudotuberculosis L-asparaginase was the most immunogenic, E. coli--the least immunogenic. E. carotovora, R. rubrum, W. succinogenes asparaginases displayed intermediate immunogenicity. The results of cross-immunogenicity evaluation have established, that blood sera of mice, that had received YpA, showed cross-immunogenicity against all the other L-asparaginase preparations except E. carotovora. During immunization with E. coli L-asparaginase the developed antibodies also bound preparation from E. carotovora. Sera from mice immunized with W. succinogenes, E. carotovora and R. rubrum L-asparaginases had cross-reaction only with EcA and did not react with other preparations. CONCLUSION: Cross-immunogenicity of the studied L-asparaginases was determined. A sequence of administration of the studied preparation is proposed that allows to minimize L-asparaginase neutralization by cross-reacting antibodies.


Asunto(s)
Anticuerpos Antibacterianos/sangre , Antígenos Bacterianos/inmunología , Asparaginasa/inmunología , Proteínas Bacterianas/inmunología , Animales , Especificidad de Anticuerpos , Antígenos Bacterianos/administración & dosificación , Antígenos Bacterianos/aislamiento & purificación , Asparaginasa/administración & dosificación , Asparaginasa/aislamiento & purificación , Proteínas Bacterianas/administración & dosificación , Proteínas Bacterianas/aislamiento & purificación , Reacciones Cruzadas , Escherichia coli/química , Escherichia coli/enzimología , Sueros Inmunes , Ratones , Ratones Endogámicos C57BL , Pectobacterium carotovorum/química , Pectobacterium carotovorum/enzimología , Rhodospirillum rubrum/química , Rhodospirillum rubrum/enzimología , Wolinella/química , Wolinella/enzimología , Yersinia pseudotuberculosis/química , Yersinia pseudotuberculosis/enzimología
19.
Biomed Khim ; 59(2): 192-208, 2013.
Artículo en Ruso | MEDLINE | ID: mdl-23789346

RESUMEN

The recombinant producer of Rhodospirillum rubrum L-asparaginase (RrA) was received and purification procedure of RrA was developed. It was shown that RrA has following biochemical and catalytic characteristics: K(m) for L-asn 0.22 MM, pH optimum 9.2; temperature optimum 54 degrees C; pI = 5.1 +/- 0.3; L-gln activity seems to be low-to-negligible. K562, DU145 and MDA-MB-231 cellular lines displayed significant sensitivity towards the enzyme (IC50 = 1.80; 9.19 and 34.62 ME/ml, respectively. In comparison with L-asparaginases from E. coli II type (EcA) and Erwinia carotovora (EwA) cytotoxicity of RrA seems to be higher than EwA, but lower than EcA. 10-fold i.p. RrA administration (4000 ME/kg per day) in L5178y bearing mice showed T/C = 172%. The received results show that RrA belongs to I type cellular L-asparaginases with low L-gln activity and the high antiproliferative effect.


Asunto(s)
Antineoplásicos/farmacología , Asparaginasa/farmacología , Proteínas Bacterianas/farmacología , Proliferación Celular/efectos de los fármacos , Glutaminasa/farmacología , Rhodospirillum rubrum/enzimología , Animales , Antineoplásicos/química , Antineoplásicos/metabolismo , Asparaginasa/biosíntesis , Asparaginasa/química , Asparaginasa/genética , Asparaginasa/aislamiento & purificación , Proteínas Bacterianas/biosíntesis , Proteínas Bacterianas/química , Proteínas Bacterianas/genética , Proteínas Bacterianas/aislamiento & purificación , Ensayos de Selección de Medicamentos Antitumorales , Glutaminasa/biosíntesis , Glutaminasa/química , Glutaminasa/genética , Glutaminasa/aislamiento & purificación , Humanos , Células K562 , Ratones , Proteínas Recombinantes/biosíntesis , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/aislamiento & purificación , Proteínas Recombinantes/farmacología , Rhodospirillum rubrum/genética
20.
Prikl Biokhim Mikrobiol ; 49(1): 24-8, 2013.
Artículo en Ruso | MEDLINE | ID: mdl-23662446

RESUMEN

The physicochemical, catalytic, and antiproliferative activity of a recombinant L-asparaginase from Yersinia pseudotuberculosis (YpA) have been studied. The following results were obtained: the K(M) value for L-asparagine is 17 +/- 0.9 microM, the optimal temperature is 60 degrees C, pH is 8.0, pI is 5.4 +/- 0.3, the L-glutaminase activity is no more than 5-6% of the L-asparaginase activity, and the antiproliferative activity on the Fisher L5178y lymphadenosis cell line comprised T/C = 136% (p < 0.001) at a 15% recovery rate. The described characteristic allows one to regard YpA as an antitumor enzyme with biological features similar to the L-asparaginase of E. coli.


Asunto(s)
Antineoplásicos , Asparaginasa , Proteínas Bacterianas , Proliferación Celular/efectos de los fármacos , Neoplasias Experimentales , Yersinia pseudotuberculosis/enzimología , Animales , Antineoplásicos/química , Antineoplásicos/aislamiento & purificación , Antineoplásicos/farmacología , Asparaginasa/química , Asparaginasa/aislamiento & purificación , Asparaginasa/farmacología , Proteínas Bacterianas/química , Proteínas Bacterianas/aislamiento & purificación , Proteínas Bacterianas/farmacología , Femenino , Ratones , Ratones Endogámicos DBA , Neoplasias Experimentales/tratamiento farmacológico , Neoplasias Experimentales/metabolismo , Neoplasias Experimentales/patología
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