Optimization of the fusion protein rhIL7-BAPmut renaturation process from the Escherichia coli inclusion bodies and its practical application

  • M. O. Usenko Institute of Molecular Biology and Genetics of Natl. Acad. Sci. of Ukraine, Ukraine, 03143, Kyiv, Akad. Zabolotnogo str., 150; State Institute of Genetic and Regenerative Medicine NAMS of Ukraine, Ukraine, 04114, Kyiv, Vyshgorodska str., 67
  • O. V. Okunev Institute of Molecular Biology and Genetics of Natl. Acad. Sci. of Ukraine, Ukraine, 03143, Kyiv, Akad. Zabolotnogo str., 150; State Institute of Genetic and Regenerative Medicine NAMS of Ukraine, Ukraine, 04114, Kyiv, Vyshgorodska str., 67
  • K. I. Bentsionova State Institution “Soils Protection Institute of Ukraine”, Ukraine, 03190, Kyiv, Babushkina str., 3
  • O. B. Gorbatiuk Institute of Molecular Biology and Genetics NAS of Ukraine, Ukraine, 03143, Kyiv, Zabolotnogo str., 150; State Institute of Genetic and Regenerative Medicine NAMS of Ukraine, Ukraine, 04114, Kyiv, Vyshgorodska str., 67
  • D. M. Irodov Institute of Molecular Biology and Genetics NAS of Ukraine, Ukraine, 03143, Kyiv, Zabolotnogo str., 150; State Institute of Genetic and Regenerative Medicine NAMS of Ukraine, Ukraine, 04114, Kyiv, Vyshgorodska str., 67
  • M. V. Koval’chuk Institute of Molecular Biology and Genetics NAS of Ukraine, Ukraine, 03143, Kyiv, Zabolotnogo str., 150; State Institute of Genetic and Regenerative Medicine NAMS of Ukraine, Ukraine, 04114, Kyiv, Vyshgorodska str., 67
  • V. A. Kordium Institute of Molecular Biology and Genetics NAS of Ukraine, Ukraine, 03143, Kyiv, Zabolotnogo str., 150; State Institute of Genetic and Regenerative Medicine NAMS of Ukraine, Ukraine, 04114, Kyiv, Vyshgorodska str., 67
DOI: https://doi.org/10.7124/visnyk.utgis.17.1.1199

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.
Keywords: IL-7, BAPmut, inclusion bodies, renaturation.

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