A novel secreted metzincin metalloproteinase from Bacillus intermedius

The mprBi gene from Bacillus intermedius 3-19 encoding a novel secreted metalloproteinase was identified. The mpriBi gene was expressed in an extracellular proteinase-deficient Bacillus subtilis BG 2036 strain and the corresponding protein was characterized biochemically. The 19. kDa MprBi protein was purified to homogeneity and sequenced by mass spectroscopy and Edman degradation methods. Amino acid sequence analysis of MprBi identified an active site motif HEYGHNFGLPHD and a conserved structural component Met-turn, both of which are unique features of the metzincin clan. Furthermore, MprBi harbors a number of distinct sequence elements characteristic of proteinase domains in eukaryotic adamalysins. We conclude that MprBi and similar proteins from other Bacillus species form a novel group of metzincin metalloproteinases in prokaryotes. © 2010 Federation of European Biochemical Societies

The expression of the serine proteinase gene of Bacillus intermedius in Bacillus subtilis

The gene encoding for Bacillus intermedius serine proteinase was cloned and the complete nucleotide sequence was determined. Gene expression was explored in the protease-deficient strain Bacillus subtilis AJ73 during different stages of growth. Catabolite repression involved in control of proteinase expression during transition state and onset of sporulation was not efficient at the late stationary phase. Salt stress leads to induction of serine proteinase production during B. subtilis AJ73(pCS9) post-exponential growth. Expression of proteinase in B. subtilis deg-mutants may be controlled by DegU regulator. B. subtilis spo0-mutants failed to accomplish B. intermedius proteinase production. These data suggest complex network regulation of B. intermedius serine proteinase expression, including the action of spo0, degU, catabolite repression and demonstrate changes in control of enzyme biosynthesis at different stages of growth. © 2006 Elsevier GmbH. All rights reserved

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

Glutamyl Endopeptidases: The Puzzle of Substrate Specificity

Glutamyl endopeptidases (GEPases) are chymotrypsin-like enzymes that preferentially cleave the peptide bonds of the -carboxyl groups of glutamic acid. Despite the many years of research, the structural determinants underlying the strong substrate specificity of GEPases still remain unclear. In this review, data concerning the molecular mechanisms that determine the substrate preference of GEPases is generalized. In addition, the biological functions of and modern trends in the research into these enzymes are outlined.</jats:p

Application of the vacuum casting technology in the production of small-size gas turbine engine blade machines

The application of casting technologies in the production of parts and assemblies of small-size gas turbine engines is justified in the paper. The technology of vacuum casting in gypsum molds was tested during the production of an experimental centrifugal compressor of a small-size gas turbine engine. On the basis of a 3D model of the designed centrifugal compressor, computational studies of vacuum casting were carried out and rational parameters of the technological process were determined. Prototypes of the developed centrifugal compressor of a small-size gas turbine engine were made. The results of calculations and the performed technological experiment confirmed the fill rate of the gating form and the absence of short pour. The distribution of shrinkage porosity and cavities corresponds to the design values and is concentrated in the central part of the casting that is subjected to subsequent machining. The area of the blades, disc and sleeve is formed without defects. The use of casting technologies in the production of parts and assemblies of small-size gas turbine engines assures the required quality with a comparatively low price of the finished product, making it possible to achieve the balance between the cost of the technology and the quality of the product made according to this technology.</jats:p

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

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