Engineering of recombinant Staphylococcal protein A with cysteine residue (SPA-Cys) for preparation of affi nity chromatography stationary phase and formation of bioselective element of immunosensor. Methods. DNA sequences encoding IgG-binding region of SPA, His-tag and cysteine were genetically fused and expressed in E. coli. SPA-Cys was immobilized on maleimide-functionalized silica beads for affi nity chromatography stationary phase preparation and on a gold sensor surface as a bioselective element of immunosensor. Results. SPA-Cys was expressed at a high-level in a soluble form. The target protein was purifi ed and showed a high IgG-binding activity. The capacity of the obtained SPA-Cys-based affi nity chromatography stationary phase was 10-12 mg of IgG /ml. The purity of eluted IgG was more than 95 % in one-step purifi cation procedure. The developed SPA-Cys-based bioselective element of immunosensor selectively interacted with human IgG and did not interact with the control proteins. Conclusions. The recombinant Staphylococcal protein A with cysteine residue was successfully used for the preparation of affi nity chromatography stationary phase and formation of the bioselective element of immunosensor. K e y w o r d s: antibodies, recombinant Staphylococcal protein A, protein immobilization, affi nity chromatography, immunosensor, surface plasmon resonance.
Protein splicing is a post-translational autocatalystic excision of internal protein sequence (intein) with the subsequent ligation of the flanking polypeptides (exteins). The high specificity of excision ensured by intein makes it possible to use a phenomenon of protein splicing for the biotechnology purposes. The aim of this work was optimization of obtaining and purification of the recombinant human growth hormone using the protein splicing. It was experimentally demonstrated that the use of modified intein as auto-removal affine marker makes it possible to perform the rapid and cheap isolation of the recombinant protein Hgh.Furthermore, this approach allows to obtain the human growth hormone with native Nterminus, without formyl-metionine.Protein splicing is a post-translational autocatalystic excision of internal protein sequence (intein) with the subsequent ligation of the flanking polypeptides (exteins). The high
As a result of immunological studies of the last 25 years it turned out that interleukin-7 (IL-7) is one of the most important regulatory cytokines of the immune system. It is special in many concerns. Having a strong impact on the development, proliferation and activation of immune cells it is, however, cannot be attributed to the classic cytokine-activators, like IL-2, as many types of cells require its presence at almost all stages of the development, and its spectrum of activities is impressive. The review aims to summarise the numerous data obtained on the IL-7 structure, biological activity, biological significance and features of functioning studied both in vitro and in vivo. We made the main emphasis on the IL-7 signal transduction pathways that determine the fate of T and B cells, as well as some other cells important for homeostasis of the organism. In our opinion, the analysis of literature in this field not only reveals a host of questions still unanswered, but also makes the path for perspective investigation more visible.
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