[Identification of Clinical Isolates of the Bacillus cereus Group and Their Characterization by Mass Spectrometry and Electron Microscopy].

Bacillus cereus is a spore-forming bacterium found in the environment mainly in soil. Bacillus spores are known to be extremely resistant not only to environmental factors, but also to various sanitation regimes. This leads to spore contamination of toxin-producing strains in hospital and food equipment and, therefore, poses a great threat to human health. Two clinical isolates identified as B. cereus and B. cytotoxicus were used in the present work. It was shown that their calcium ion content was significantly lower than that of the reference strains. According to electron microscopy, one of the SRCC 19/16 isolates has an enlarged exosporium, and the SRCC 1208 isolate has large electron-dense inclusions of an unclear nature during sporulation. We can assume that these contain a biologically active component with a cytotoxic effect and possibly play a role in pathogenesis. Comparative chemical, biochemical, physiological, and ultrastructural analysis of spores of clinical isolates and reference strains of B. cereus was performed. The results we obtained deepen our understanding of the properties of spores that contribute to the increased pathogenicity of B. cereus group species.

Recombinant Human Erythropoietin with Additional Processable Protein Domains: Purification of Protein Synthesized in Escherichia coli Heterologous Expression System.

Three variants of human recombinant erythropoietin (rhEPO) with additional N-terminal protein domains were obtained by synthesis in an Escherichia coli heterologous expression system. These domains included (i) maltose-binding protein (MBP), (ii) MBP with six histidine residues (6His) in N-terminal position, (iii) s-tag (15-a.a. oligopeptide derived from bovine pancreatic ribonuclease A) with N-terminal 6His. Both variants of the chimeric protein containing MBP domain were prone to aggregation under nondenaturing conditions, and further purification of EPO after the domain cleavage by enterokinase proved to be impossible. In the case of 6His-s-tag-EPO chimeric protein, the products obtained after cleavage with enterokinase were successfully separated by column chromatography, and rhEPO without additional domains was obtained. Results of MALDI-TOF mass spectrometry showed that after refolding 6His-s-tag-EPO formed a structure similar to that of one of native EPO with two disulfide bonds. Both 6His-s-tag-EPO and rhEPO without additional protein domains purified after proteolysis possessed the same biological activity in vitro in the cell culture.