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1.
Biomed Khim ; 69(1): 19-38, 2023 Feb.
Article in Russian | MEDLINE | ID: mdl-36857424

ABSTRACT

L-asparaginase (EC 3.5.1.1) is one of the most demanded enzymes used in the pharmaceutical industry as a drug and in the food industry to prevent the formation of toxic acrylamide. Researchers aimed to improve specific activity and reduce side effects to create safer and more potent enzyme products. However, protein modifications and heterologous expression remain problematic in the production of asparaginases from different species. Heterologous expression in optimized producer strains is rationally organized; therefore, modified and heterologous protein expression is enhanced, which is the main strategy in the production of asparaginase. This strategy solves several problems: incorrect protein folding, metabolic load on the producer strain and codon misreading, which affects translation and final protein domains, leading to a decrease in catalytic activity. The main approaches developed to improve the heterologous expression of L-asparaginases are considered in this paper.


Subject(s)
Acrylamide , Asparaginase , Protein Processing, Post-Translational
2.
Bull Exp Biol Med ; 167(3): 375-379, 2019 Jul.
Article in English | MEDLINE | ID: mdl-31346881

ABSTRACT

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.


Subject(s)
Bacteria/genetics , Bacterial Proteins/biosynthesis , Escherichia coli/metabolism , Methyl-Accepting Chemotaxis Proteins/biosynthesis , Bacterial Proteins/genetics , Bioreactors/microbiology , Chemotaxis/genetics , Escherichia coli/genetics , Escherichia coli Proteins , Gene Expression/genetics , Methyl-Accepting Chemotaxis Proteins/genetics , Thermotoga
3.
Bull Exp Biol Med ; 165(4): 461-464, 2018 Aug.
Article in English | MEDLINE | ID: mdl-30121908

ABSTRACT

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.


Subject(s)
Amino Acids/chemistry , Amino Acids/metabolism , Culture Media/chemistry , Recombinant Proteins/chemistry , Recombinant Proteins/metabolism , Fibroblasts/metabolism , Humans , Hydrogen-Ion Concentration , Temperature
4.
Bull Exp Biol Med ; 164(4): 478-482, 2018 Mar.
Article in English | MEDLINE | ID: mdl-29504103

ABSTRACT

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.


Subject(s)
Alternative Splicing , B-Lymphocytes/immunology , CD4-Positive T-Lymphocytes/immunology , CD8-Positive T-Lymphocytes/immunology , Endonucleases/genetics , Killer Cells, Natural/immunology , Telomerase/genetics , Apoptosis/genetics , Apoptosis/immunology , B-Lymphocytes/cytology , CD4-Positive T-Lymphocytes/cytology , CD8-Positive T-Lymphocytes/cytology , Endonucleases/immunology , Gene Expression Regulation, Enzymologic , Genes, Reporter , Green Fluorescent Proteins/genetics , Green Fluorescent Proteins/metabolism , Humans , Immunophenotyping , Killer Cells, Natural/cytology , Signal Transduction , Telomerase/immunology
5.
Biomed Khim ; 63(4): 296-305, 2017 Jul.
Article in Russian | MEDLINE | ID: mdl-28862599

ABSTRACT

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.


Subject(s)
Alternative Splicing , CD4-Positive T-Lymphocytes/enzymology , CD8-Positive T-Lymphocytes/enzymology , DNA Damage , Endodeoxyribonucleases/metabolism , Telomerase/genetics , Cells, Cultured , Cisplatin , Humans
6.
Vopr Pitan ; 86(3): 19-31, 2017.
Article in Russian | MEDLINE | ID: mdl-30645859

ABSTRACT

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.

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