Optimization of the fusion protein rhIL7-BAPmut renaturation process from the Escherichia coli inclusion bodies and its practical application
Journal Title: Вісник Українського товариства генетиків і селекціонерів - Year 2019, Vol 17, Issue 1
Abstract
Aim. The aim of our work was to optimize the renaturation method of the rhIL7-BAPmut fusion protein based on recombinant human interleukin-7 (rhIL7) and bacterial alkaline phosphatase with enhanced catalytic properties (BAPmut) for its obtaining in functionally active form. Methods. The cells of E. coli strain BL21(DE3) were transformed with pET24-IL7-BAPmut plasmid vector. Protein synthesis was induced by autoinduction protocol. Immobilized-metal affinity chromatography (IMAС) and slow dilution methods were applied for rhIL7-BAPmut fusion protein renaturation from bacterial inclusion bodies in vitro. Results. Combination of IMAС method and slow dilution at the presence of arginine, GSH/ GSSG and Mg2+ ions provided obtaining of rhIL7-BAPmut in pure and active form. Bifunctional activity of rhIL7-BAPmut after refolding is confirmed immunochemically by binding with specific antibodies. Conclusions. It was shown that application of rhIL7-BAPmut allows to reduce the time of the screening of immune combinatory libraries of variable genes of IgG and does not require specific primary and secondary antibodies. The rhIL7-BAPmut fusion protein also can be used for qualitative and quantitative analysis of IL-7 receptors.
Authors and Affiliations
M. O. Usenko, O. V. Okunev, K. I. Bentsionova, O. B. Gorbatiuk, D. M. Irodov, M. V. Koval’chuk, V. A. Kordium
Keywords
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- EP ID EP660051
- DOI 10.7124/visnyk.utgis.17.1.1199
- Views 95
- Downloads 0
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