Получение рекомбинантных нейротрофинов человека для биомедицинских исследований

The genes of nerve growth factor (NGF), brain neurotrophic factor (BDNF) and human neurotrophic factor 3 (NT-3) were cloned and expressed in Escherichia coli. Methods of purification and renaturation of the proneurotrophins were developed. It was shown that the recombinant pro-NGF, pro-BDNF and pro-NT-3 induce a differentiation of chicken dorsal root ganglia cell culture.Клонированы гены фактора роста нервов (NGF), нейротрофического фактора головного мозга (BDNF) и нейротрофического фактора 3 (NT-3) человека и осуществлена их экспрессия в клетках Escherichia coli. Разработаны схемы очистки и ренатурации соответствующих пробелков. Продемонстрировано, что рекомбинантные про-NGF, про-BDNF, про-NT-3 оказывают дифференцирующее действие на культуру спинномозговых ганглиев эмбрионов кур

Production of human recombinant neurotrophins for biomedical studies

The genes of nerve growth factor (NGF), brain neurotrophic factor (BDNF) and human neurotrophic factor 3 (NT-3) were cloned and expressed in Escherichia coli. Methods of purification and renaturation of the proneurotrophins were developed. It was shown that the recombinant pro-NGF, pro-BDNF and pro-NT-3 induce a differentiation of chicken dorsal root ganglia cell culture

Protealysin is not Secreted Constitutively

© 2019 Bentham Science Publishers. Background: Protealysin, a zinc metalloprotease of Serratia proteamaculans, is the prototype of a new group within the peptidase family M4. Protealysin-like proteases (PLPs) are widely spread in bacteria but are also found in fungi and archaea. The biological functions of PLPs have not been well studied, but published data showed the involvement of enzymes of this group in the interaction of bacteria with higher organisms, and most likely in the pathogenesis. Such functionality requires the release of the proteases from bacterial cells; however, the data on the cellular localization of PLPs are contradictory and no direct data of this kind have been published. Objective: Here, the protealysin cellular localization was studied for the first time using immunochemical methods. Methods and Results: We have produced polyclonal rabbit antibodies against the protealysin precursor. The enzyme was evaluated in cells and medium of periodic culture of S. proteamaculans 94 using Western blotting as well as the enzyme localization was analysed by immunoelectron microscopy. It was shown that more than 99% of the enzyme is in a cell-associated form. Protealysin is accumulated in cells as an inactive precursor. It matures only after the release from cells (after their lysis). Immunoelectron microscopy analysis of bacterial cells has revealed no specific localization of protealysin; it was evenly distributed in the cytoplasm. Conclusion: The data obtained suggest that S. proteamaculans protealysin and supposedly other protealysin-like proteases are not secreted constitutively and their release from bacteria is likely induced by a certain stimulus such as a contact with a eukaryotic cell. This finding is critical for further studies of the involvement of these enzymes in pathogenesis

Efficiency of Promoters of Human Genes <i>FAP</i> and <i>CTGF</i> at Organism Level in a <i>Danio rerio</i> Model

The identification of tissue-specific promoters for gene therapeutic constructs is one of the aims of complex tumor therapy. The genes encoding the fibroblast activation protein (FAP) and the connective tissue growth factor (CTGF) can function in tumor-associated stromal cells but are practically inactive in normal adult cells. Accordingly, the promoters of these genes can be used to develop vectors targeted to the tumor microenvironment. However, the efficiency of these promoters within genetic constructs remains underexplored, particularly, at the organism level. Here, we used the model of Danio rerio embryos to study the efficiency of transient expression of marker genes under the control of promoters of the FAP, CTGF, and immediate early genes of Human cytomegalovirus (CMV). Within 96 h after the injection of vectors, the CTGF and CMV promoters provided similar equal efficiency of reporter protein accumulation. In the case of the FAP promoter, a high level of reporter protein accumulation was observed only in certain zebrafish individuals that were considered developmentally abnormal. Disturbed embryogenesis was the factor of changes in the exogenous FAP promoter function. The data obtained make a significant contribution to understanding the function of the human CTGF and FAP promoters within vectors to assess their potential in gene therapy

Functional efficiency of PCR vectors in vitro and at the organism level.

The functional efficiency of the expression cassettes integrated into a plasmid and a PCR- amplified fragment was comparatively analyzed after transient transfection in vitro or introduction into the developing embryo of Danio rerio. The cassettes contained the reporter genes, luciferase of Photinus pyralis (luc) or enhanced green fluorescent protein, under the control of the promoter of human cytomegalovirus immediate-early genes. In the in vitro system, the efficiency of the circular plasmid was 2.5 times higher than that of the PCR- amplified fragment. The effect of mutations in the expression cassette on the efficiency of the transgene expression in the PCR- amplified fragment was quantitatively evaluated. The mutations generated after 25 amplification cycles with Taq DNA polymerase decreased luciferase activity in transfected cells by 65-85%. Thus, mutations are the key factor of decreased functional efficiency of the PCR- amplified fragment relative to the circular plasmid in this experimental model, while other factors apparently have a lesser impact. At the organism level, no significant difference in the expression efficiency of the plasmid and PCR- amplified fragment has been revealed. Comparison of the vector efficiencies in in vivo and in vitro systems demonstrates that the level of luciferase in the D. rerio cell lysate, normalized to the molar concentration of the vector, is by three orders of magnitude higher than that after the cell transfection in vitro, which indicates that the quantitative data obtained for in vitro systems should not be directly extrapolated to the organism level