Production and analysis of the biological properties of recombinant human granulocyte colony stimulating factor chimeric form

The aim of this work was to design and study biological properties of the recombinant human granulocyte colony stimulating factor (G-CSF), «linked» to apolipoprotein A-I (apoA-I) by a peptide linker, for obtaining in perspective a prolong form of the drug based on this cytokine.Material and methods. The nucleotide sequences of the genes encoding G-CSF and apoA-I were designed and optimized for expression in Pichia pastoris yeast using several computer programs. The assembly of the gene coding for the G-CSF-apoA-I chimeric cytokine, its cloning in the pPICZa-A vector, and expression in P. pastoris cells were performed using standard genetic engineering methods. Purification of the chimeric cytokine was carried out by two-stage ion-exchange chromatography. The biological activity of the chimera was determined in vitro on rat and human bone marrow cells (BMC) using flow cytometry, cell cycle analysis and myelograms.Results. A recombinant P pastoris X-33 yeast strain producing a chimeric cytokine containing the amino acid sequence G-CSF from the N-terminus, and mature human apoA-I from the C-terminus was constructed. In experiments on BMC of rat, it was shown that G-CSF-apoA-I increases the number of granulocytes in 1.8-2 times less compared with G-CSF. At the same time, the chimeric cytokine maintained the viability of monocytic and lymphocytic cells. Unlike G-CSF, the chimera increased the number of blast cells and normalized neutrophil segmentation, reducing the number of anomalies 1.5 times more efficiently.Conclusion. A new chimeric cytokine G-CSF-apoA-I was constructed, exhibiting the properties of not only a colony-stimulating factor, but also a growth factor, supporting the viability of other types of BMC

Creation of a recombinant Komagataella phaffii strain, a producer of proteinase K from Tritirachium album

The objects of the study were recombinant clones of Komagataella phaffii K51 carrying the heterologous proteinase K (PK-w) gene from Tritirachium album integrated into their genome as well as samples of recombinant proteinase K isolated from these clones. The aims of this work were i) to determine whether it is possible to create recombinant K. phaffii K51 clones overexpressing functionally active proteinase K from T. album and ii) to analyze the enzymatic activity of the resulting recombinant enzyme. The following methods were used: computational analysis of primary structure of the proteinase K gene, molecular biological methods (PCR, electrophoresis of DNA in an agarose gel, electrophoresis of proteins in an SDS polyacrylamide gel under denaturing conditions, spectrophotometry, and quantitative assays of protease activity), and genetic engineering techniques (cloning and selection of genes in bacterial cells Escherichia coli TOP10 and in the methylotrophic yeast K. phaffii K51). The gene encoding natural proteinase K (PK-w) was designed and optimized for expression in K. phaffii K51. The proteinase K gene was synthesized and cloned within the plasmid pPICZα-A vector in E. coli TOP10 cells. The proteinase K gene was inserted into pPICZα-A in such a way that – at a subsequent stage of transfection into yeast cells – it was efficiently expressed under the control of the promoter and terminator of the AOX1 gene, and the product of the exogenous gene contained the signal peptide of the Saccharomyces cerevisiae a-factor to ensure the protein’s secretion into the culture medium. The resultant recombinant plasmid (pPICZα-A/PK-w) was transfected into K. phaffii K51 cells. A recombinant K. phaffii K51 clone was obtained that carried the synthetic proteinase K gene and ensured its effective expression and secretion into the culture medium. An approximate productivity of the yeast recombinant clones for recombinant proteinase K was 25 μg/ mL after 4 days of cultivation. The resulting recombinant protease has a high specific proteolytic activity: ~5000 U/mg

Rivaroxaban with or without aspirin in stable cardiovascular disease

BACKGROUND: We evaluated whether rivaroxaban alone or in combination with aspirin would be more effective than aspirin alone for secondary cardiovascular prevention. METHODS: In this double-blind trial, we randomly assigned 27,395 participants with stable atherosclerotic vascular disease to receive rivaroxaban (2.5 mg twice daily) plus aspirin (100 mg once daily), rivaroxaban (5 mg twice daily), or aspirin (100 mg once daily). The primary outcome was a composite of cardiovascular death, stroke, or myocardial infarction. The study was stopped for superiority of the rivaroxaban-plus-aspirin group after a mean follow-up of 23 months. RESULTS: The primary outcome occurred in fewer patients in the rivaroxaban-plus-aspirin group than in the aspirin-alone group (379 patients [4.1%] vs. 496 patients [5.4%]; hazard ratio, 0.76; 95% confidence interval [CI], 0.66 to 0.86; P<0.001; z=−4.126), but major bleeding events occurred in more patients in the rivaroxaban-plus-aspirin group (288 patients [3.1%] vs. 170 patients [1.9%]; hazard ratio, 1.70; 95% CI, 1.40 to 2.05; P<0.001). There was no significant difference in intracranial or fatal bleeding between these two groups. There were 313 deaths (3.4%) in the rivaroxaban-plus-aspirin group as compared with 378 (4.1%) in the aspirin-alone group (hazard ratio, 0.82; 95% CI, 0.71 to 0.96; P=0.01; threshold P value for significance, 0.0025). The primary outcome did not occur in significantly fewer patients in the rivaroxaban-alone group than in the aspirin-alone group, but major bleeding events occurred in more patients in the rivaroxaban-alone group. CONCLUSIONS: Among patients with stable atherosclerotic vascular disease, those assigned to rivaroxaban (2.5 mg twice daily) plus aspirin had better cardiovascular outcomes and more major bleeding events than those assigned to aspirin alone. Rivaroxaban (5 mg twice daily) alone did not result in better cardiovascular outcomes than aspirin alone and resulted in more major bleeding events