Obtaining of bifunctional fusion proteins based on human interleukin 7 and their application for biomedical research

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ICE Krakow

ICE Krakow

ul. Marii Konopnickiej 17 30-302 Kraków


Gorbatiuk, Oksana (Institute of Molecular Biology and Genetics of NASU)


Interleukin 7 (IL-7) is an immune cytokine that plays a critical role in the maturation and homeostasis of T and B-lymphocytes. The absence of IL-7 significantly affects the proliferation of early lymphoid progenitor cells and leads to the development of severe combined immunodeficiency syndrome. To date, there are active studies of recombinant IL-7 as a means of restoring the immune system of people who have undergone bone marrow transplantation, highly active antiretroviral therapy and chemotherapy. New experimental data are concentrated on the application of IL-7 in the treatment of COVID-19 and as an adjuvant in during vaccination process. IL-7 is widely studied as a therapeutic agent for various immune disorders, that why biomarkers related to immune cell responses to IL-7 that can be used to monitor immune status, individual pharmacological responses to IL-7 treatment, are needed. IL-7 activity can be tested using peripheral blood mononuclear cells (PBMCs) from different donors. The individual response of human PBMCs to IL-7 is valuable prognostic information about the state of the immune system. This information is useful for further large-scale study of the response of T-cell subgroups to IL-7 therapy. This investigations requires highly purified IL-7. The work aimed to obtain rhIL-7 in Escherichia coli and allow protein renaturation with further testing of peripheral blood mononuclear cells (PBMCs) responsiveness to highly purified cytokine. The DNA sequence encoding human IL-7 was subcloned into a pET24a(+) expression vector under control of the T7 promoter and upstream of the vector-derived 6xHis-tag. pET24-hIL7 was transformed into E. coli, protein synthesis was induced with the auto-induction protocol. Immobilized metal affinity chromatography was used for the purification of solubilized protein. Subsequent refolding protocols of rhIL7-His were developed using dilution, gel filtration, on-column refolding, and dialysis strategies. The highest renaturation efficiency of rhIL7-His was obtained in the dialysis method. Gel filtration and refolding on metal affinity sorbent showed comparable lower efficiencies. The activity of rhIL7-His renaturated by dialysis and gel filtration was tested on PBMSc. The response of phytohemagglutinin activated lymphocytes of human peripheral blood was measured in the MTT test. Analysis shows the comparable activity of standard rhIL7 and rhIL7-His renaturated by dialysis. It can be concluded that after dialysis the rhIL7-His protein is obtained in a fully functional form. The study revealed that responsiveness to IL-7 is various for the PBMCs originated from different individuals. Our research assumes that the personalized responsiveness of human PBMCs to IL-7 may be considered as valuable prognostic information about the immune system state and the T cells response to IL-7-based therapies.

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