Open AccessObtaining of the recombinant rhIL7-BAPmut fusion protein and its functional characterization
Author(s) -
M. O. Usenko,
O. V. Okunev,
K. I. Bentsionova,
O. B. Gorbatiuk,
Д. М. Іродов,
В. А. Кордюм
Publication year - 2019
Publication title -
faktori eksperimentalʹnoï evolûcìï organìzmìv
Language(s) - English
Resource type - Journals
eISSN - 2415-3826
pISSN - 2219-3782
DOI - 10.7124/feeo.v25.1185
Subject(s) - fusion protein , recombinant dna , inclusion bodies , lac operon , complementary dna , microbiology and biotechnology , plasmid , target protein , protein a/g , antibody , biology , expression vector , biochemistry , chemistry , gene , genetics
Aim. The purpose was to obtain a functionally active form of the recombinant fusion protein based on the human interleukin-7 (rhIL7) and bacterial alkaline phosphatase (BAPmut). Methods. The DNA sequences encoding rhIL-7 and BAPmut were subcloned into the pET24a(+) plasmid vector containing the 6His-tag sequence for further chromatographic purification of the target protein. The cells of E. coli strain BL21(DE3) were transformed with pET24-rhIL7-BAPmut plasmid vector. Protein synthesis was induced by IPTG and by autoinduction. Bifunctional activity of rhIL7-BAPmut after refolding via dilution is confirmed immunochemically by binding to specific antibodies. Results. RhIL7-BAPmut protein has been designed. It was shown that autoinduction protocol provides a significantly higher level of protein synthesis in E. coli compared with IPTG induction. In vitro method of purification and renaturation of rhIL7-BAPmut from inclusion bodies has been worked out. It has been shown that rhIL7-BAPmut has a specific biological activity after renaturation. Conclusions. The obtained rhIL7-BAPmut protein can be used for screening of immune combinatorial cDNA libraries of immunoglobulins variable genes, that are the sources of specific single chain antibodies. The protein also can be used for qualitative and quantitative analysis of IL-7 receptors.
Keywords: IL-7, BAPmut, inclusion bodies, renaturation.