Obtaining of the recombinant rhIL7-BAPmut fusion protein and its functional characterization

  • M. O. Usenko
  • O. V. Okunev
  • K. I. Bentsionova
  • O. B. Gorbatiuk
  • D. M. Irodov
  • V. A. Kordium
DOI: https://doi.org/10.7124/FEEO.v25.1185

Abstract

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.

References

Takada K., Jameson S.C. Naive T cell homeostasis: from awareness of space to a sense of place. Nat. Rev. Immun. 2009. Vol. 9. P. 823–832. doi: 10.1038/nri2657.

Corfe S.A., Paige C.J. The many roles of IL-7 in B cell development; Mediator of survival, proliferation and differentiation. Seminars in Immunology. 2012. Vol. 24. P. 198–208. doi: 10.1016/j.smim.2012.02.001.

Slyvka A.V., Okunev O.V. Molecular mechanisms of versatile biological activity of interleukin-7. Biopolym. Cell. 2014. Vol. 30. P. 349–357. doi: 10.7124/bc.0008B1.

Palmer M.J., Mahajan V.S., Trajman L.C., Irvine D.J., Lauffenburger D.A., Chen J. Interleukin-7 Receptor Signaling Network: An Integrated Systems Perspective. Cellular & Molecular Immunology. 2008. Vol. 5. P. 79–89. doi: 10.1038/cmi.2008.10.

Lundström W., Fewke N.M., Mackall C.L. IL-7 in human health and disease. Seminars in immunology. 2012. Vol. 24. P. 218–224. doi: 10.1016/j.smim.2012.02.005.

Hartling H.J., Ryder L.P., Ullum H., Ødum N., Nielsen S.D. Gene variation in IL-7 receptor (IL-7R)α affects IL-7R response in CD4+ T cells in HIV-infected individuals. Sci. reports. 2017. Vol. 7. P. 42036 (1–8). doi: 10.1038/srep42036.

Haas J., Korporal M., Schwarz A., Balint B., Wildemann B. The interleukin-7 receptor α chain contributes to altered homeo-stasis of regulatory T cells in multiple sclerosis. Eur. J. Immunol. 2011. Vol. 41. P. 845–853. doi: 10.1002/eji.201041139.

Taheri M., Sayad A. Investigating the exon 6 sequence changes of interleukin 7 receptor A (IL7RA) gene in patients with re-lapsing-remitting multiple sclerosis. Hum. Antibodies. 2018. Vol. 26. P. 43–48. doi: 10.3233/HAB-170320.

Lundström W., Hermanrud C., Sjöstrand M., Brauner S., WahrenHerlenius M., Olsson T., Karrenbauer V., Hillert J., Fogdell-Hahn A. Interferon beta treatment of multiple sclerosis increases serum interleukin-7. Multiple Sclerosis Journal. 2014. Vol. 20. P. 1727–1736. doi: 10.1177/1352458514532700.

Gorbatiuk O.B., Okunev O.V., Nikolaev Yu.S., Svyatenko O.V., Kordium V.A. Construction, expression, functional сharacterization and practical application of fusion protein SPA-ВAPmut. Biopolym. Cell. 2013. Vol. 29. P. 49–54. doi: 10.7124/bc.000805.

Muller B.H., Lamoure C., Le Du M.H., Cattolico L., Lajeunesse E., Lemaitre F., Pearson A., Ducancel F., Menez A., Boulain J.C. Improving Escherichia coli alkaline phosphatase efficacy by additional mutations inside and outside the catalytic pocket. Chembiochem. 2001. Vol. 2. P. 517–523. doi: 10.1002/1439-7633(20010803)2:7/8<517::aid-cbic517>3.0.co;2-h.

Sambrook J., Fritsch E. F., Maniatis T. Molecular cloning: A laboratory Manual. Second edition. New York: Cold Spring Harbor Laboratory Press, 1989. 1659 p.

Studier F.W. Protein production by auto-induction in high density shaking cultures. Protein Expr. Purif. 2005. Vol. 41. P. 207–234. doi: 10.1016/j.pep.2005.01.016.

Gorbatuk O.B., Nikolayev U.S., Irodov D.M., Dubey I.Ya., Gilchuk P.V. Refolding of ScFv-CBD fusion protein from Es-cherichia coli inclusion bodies. Biopolym. Cell. 2008. Vol. 24, № 1. Р. 51–59. [in Ukrainian]