| Abstract [eng] |
Therapeutic recombinant protein – granulocyte colony-stimulating factor (G-CSF) – is a pharmaceutical preparation widely used in medicine. G-CSF like most other drugs of low molecular weight protein origin, is characterized by a short circulation half-life in the blood, which is prolonged by various modifications of the protein molecule. Usually, after modifications, the biological activity of the protein is reduced. To solve these problems, an alternative strategy for prolonging the circulation life of the protein was chosen in this doctoral dissertation – a genetic fusion of G-CSF protein molecules via specific length and composition linkers. The first dimeric protein was constructed by fusion of two G-CSF molecules, and the second dimer by fusion of G-CSF with stem cell factor (SCF). For these proteins, synthesized by recombinant DNA technology in E. coli cells, protein purification technologies were developed, the physical and chemical properties of new homo- and heterodimers were evaluated, and their biological activity and function in vivo were determined using an animal (rat) model. In the first part of the work, it was found that the G-CSF dimer with a structured alpha helix-forming peptide linker (Lα) had the highest purity (>95%) and yield (>14%). The developed protein isolation and purification technology can be applied for small and large-scale protein production. The G-CSF homodimer had better pharmacokinetic and pharmacodynamic properties in vivo than the monomer. To demonstrate the effectiveness of the Lα-type linker in separating individual domains of the bifunctional fusion protein and having more active fusion proteins, in the second part of this work, the G-CSF and SCF heterodimers were generated. The total internal reflection ellipsometry (TIRE) data and in vitro biological studies demonstrated that the Lα linker ensures spatial separation of G-CSF and SCF monomers, allowing them to function independently. In vivo studies have shown that heterodimeric SCF-Lα-GCSF maintains a biological activity similar to that of a mixture of SCF and G-CSF monomers used in medicine. |
