Usenko M. Development of genetically engineered conjugates and their use for detection and purification of the therapeutically important proteins

The thesis considers the development of methods for obtaining bifunctional conjugates based on target proteins and marker molecules in E. coli. RhIL7-His, rhIL7-CBD and rhIL7-BAPmut-producing strains were obtained. Methods for obtaining proteins in soluble functionally active form by in vitro renaturation have been optimized. The rhIL7-CBD fusion protein, after immobilization on microcrystalline cellulose CC31, and rhIL7-His, after immobilization on a metal-affinity media, were used to produce polyclonal antibodies specific for human interleukin 7 with a purity of more than 95%. Using rhIL7-BAPmut a combinatorial cDNA library of immunoglobulin variable genes was screened. E. coli strains producers of scFv(IFNβ1b)–BAPmut fusion protein and a recombinant human ASK1 protein kinase were obtained. The possibility of using the scFv(IFNβ1b)-BAPmut to detect human interferon beta-1b has been shown. The conditions of ASK1 protein kinase biosynthesis were determined, which ensured the production of an enzyme with a functional activity comparable to commercially available analogues. The variability of individual responses of human peripheral blood mononuclear cells to rhIL7 as an important prognostic indicator of the state of the immune system for further immunological studies was analyzed.