Recombinant human erythropoietin with additional processable protein domains: Purification of protein synthesized in <Emphasis Type="Italic">Escherichia coli</Emphasis> 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.     

Synthesis in <Emphasis Type="Italic">Escherichia coli</Emphasis> and Characterization of Human Recombinant Erythropoietin with Additional Heparin-Binding Domain

Recombinant human erythropoietin (EPO) with additional N-terminal heparin-binding protein domain (HBD) from bone morphogenetic protein 2 was synthesized in Escherichia coli cells. A procedure for HBD-EPO purification and refolding was developed for obtaining highly-purified HBD-EPO. The structure of recombinant HBD-EPO was close to that of the native EPO protein. HBD-EPO contained two disulfide bonds, as shown by MALDI-TOF mass spectrometry. The protein demonstrated in vitro biological activity in the proliferation of human erythroleukemia TF-1 cell test and in vivo activity in animal models. HBD-EPO increased the number of reticulocytes in the blood after subcutaneous injection and displayed local angiogenic activity after subcutaneous implantation of demineralized bone matrix (DBM) discs with immobilized HBD-EPO. We developed a quantitative sandwich ELISA method for measuring HBD-EPO concentration in solution using rabbit polyclonal serum and commercial monoclonal anti-EPO antibodies. Pharmacokinetic properties of HBD-EPO were typical for bacterially produced EPO. Under physiological conditions, HBD-EPO can reversibly bind to DBM, which is often used as an osteoplastic material for treatment of bone pathologies. The data on HBD-EPO binding to DBM and local angiogenic activity of this protein give hope for successful application of HBD-EPO immobilized on DBM in experiments on bone regeneration.     

Helicobacter pylori entry into human gastric epithelial cells: A potential determinant of virulence, persistence, and treatment failures

Background and Objectives. Intracellular location of Helicobacter pylori in human gastric epithelial cells has been observed in biopsies. Whether this reflects an ability to invade host cells and establish an intracellular niche remains to be determined. Methods. The interactions between a clinical isolate of H. pylori and primary cell cultures from human gastric epithelium or the human epithelial cell line HEp‐2 were monitored using time‐lapse photography. This technique allows studies of the dynamics of host‐microbial interactions. Results. H. pylori cells readily approached and established close contacts with epithelial cells followed by uptake of the bacteria into the cellular cytoplasm. Entry into epithelial cells was achieved through an active process of bacterial motility and penetration of the cell membranes. In conventional invasion assays using HEp‐2 cells, an increased internalization in a strain producing the vacuolating cytotoxin was observed, compared to the isogenic VacA knockout mutant. Conclusion. Invasion of gastric epithelium represents a hitherto unappreciated trait of H. pylori that could contribute to the bacterium's ability to establish persistent infection that evades the mucosal immune defense and sometimes also antimicrobial therapy. A small number of bacterial cells with a transient intracellular habitat could serve as a seeder population, providing a backup for a constantly challenged and fluctuating luminal population.     

Development of a Platform for Producing Recombinant Protein Components of Epitope Vaccines for the Prevention of COVID-19

Biochemistry (Moscow) - A new platform for creating anti-coronavirus epitope vaccines has been developed. Two loop-like epitopes with lengths of 22 and 42 amino acid residues were selected from the...     

Hyper-Resistance of the Bacillus licheniformis 24 Strain to Oxidative Stress Is Associated with Overexpression of Enzymatic Antioxidant System Genes

Molecular Biology - At the International Space Station (ISS), artificial living conditions are created and maintained to satisfy human needs, these conditions are also favorable for the growth of...     

Electrostatic binding of substituted metal phthalocyanines to enterobacterial cells: Its role in photodynamic inactivation

The effect of ionic substituents in zinc and aluminum phthalocyanine molecules and of membrane surface charge on the interaction of dyes with artificial membranes and enterobacterial cells, as well as on photosensitization efficiency was studied. It has been shown that increasing the number of positively charged substituents enhances the extent of phthalocyanine binding to Escherichia coli cells. This, along with the high quantum yield of singlet oxygen generation, determines efficient photodynamic inactivation of Gram-negative bacteria by zinc and aluminum octacationic phthalocyanines. The effect of Ca²⁺ and Mg²⁺ cations and pH on photodynamic inactivation of enterobacteria in the presence of octacationic zinc phthalocyanine has been studied. It has been shown that effects resulting in lowering negative charge on outer membrane protect bacteria against photoinactivation, which confirms the crucial role in this process of the electrostatic interaction of the photosensitizer with the cell wall. Electrostatic nature of binding is consistent with mainly electrostatic character of dye interactions with artificial membranes of different composition. Lower sensitivity of Proteus mirabilis to photodynamic inactivation, compared to that of E. coli and Salmonella enteritidis, due to low affinity of the cationic dye to the cells of this species, was found.