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

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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