Open AccessObtaining of the recombinant Rhil7-Cbd fusion protein
Author(s) -
M. O. Usenko,
O. B. Gorbatiuk,
O. V. Okunev,
Д. М. Іродов,
M. V. Kovalchuk,
В. А. Кордюм
Publication year - 2020
Publication title -
faktori eksperimentalʹnoï evolûcìï organìzmìv
Language(s) - English
Resource type - Journals
eISSN - 2415-3826
pISSN - 2219-3782
DOI - 10.7124/feeo.v26.1280
Subject(s) - inclusion bodies , fusion protein , recombinant dna , lac operon , polyclonal antibodies , microbiology and biotechnology , flag tag , target protein , expression vector , protein a/g , protein purification , biology , chemistry , biochemistry , antibody , gene , genetics
Aim. The purpose was to obtain the recombinant fusion protein based on the human interleukin-7 (rhIL7) and cellulose binding domain (CBD). Methods. The DNA sequences encoding rhIL-7 and CBD were subcloned into the pET24a(+). 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-CBD plasmid vector. Protein synthesis was induced in clones by IPTG and by autoinduction. Results. An expression cassette of rhIL7-CBD protein has been designed. Producers of rhIL7-CBD protein were obtained. It was shown that the autoinduction protocol provides protein synthesis in E. coli (but IPTG induction doesn’t). Protein was obtained in the cytoplasmic fraction in form of inclusion bodies. In vitro method of purification of rhIL7-CBD from inclusion bodies has been worked out. Conclusions. The obtained rhIL7-CBD protein can be used for the microcrystalline cellulose immunosorbent construction for the purification of the specific polyclonal IL-7 antibodies and also for qualitative and quantitative analysis of IL-7 receptors.
Keywords: IL-7, CBD, inclusion bodies, renaturation.