Development of the chromatographic medium for the affinity isolation of the recombinant hIFN-β1b based on immobilized single-chain antibodies

• DOI: 10.7124/bc.0008ec
• Publication Date: 2015-10-05T06:48:57Z
• Authors: Ia. O. Pokholenko; O. B. Gorbatiuk; O. V. Okunev; D. M. Irodov; M. I. Degtiarova; O. G. Palivoda; V. A. Kordium
• Keywords: 0301 basic medicine; 03 medical and health sciences; Molecular and Cell Biotechnologies

Aim. The development of a laboratory method for the production of immunoaffinity chromatography medium for the purification of the recombinant human IFN-??1b. Methods. A gene of the chimeric protein ScFvbINF-CBD was constructed using the DNA sequences encoding ScFv specific to hIFN-??1b and the cellulose-binding domain (CBD) of Clostridium thermocellum. The developed chimeric protein was expressed in Escherichia coli cells. The target protein was obtained in soluble, functionally active form by its renaturation from the bacterial inclusion bodies in vitro. The ScFvbINF-CBD was immobilized on a chitin carrier. Results. The introduction of CBD by gene engineering techniques enabled the oriented non-covalent immobilization of ScFvbINF-CBD on chromatographic matrix. The developed immunoaffinity medium allowed isolating the rhIFN-??1b from the complex mixture, after its renaturation from the inclusion bodies, with more than 89 % purity. Conclusion. The designed immunoaffinity medium provides isolating the rhIFN-??1b from the complex protein mixtures.<br/>????????. ?????????????????????? ???????????????????????? ?????????? ???????????????? ?????????????????????????????? ?????????????????????????????????????? ???????????????? ?????? ?????????????????? ?? ?????????????? ?????????????????????????????? IFN-??1b ????????????????. ????????????. ?????????????????????? ???????????????????????????????????? ??????, ???????????????????? ScFv ???????????????????????? ?? hIFN-??1b, ?? ???????????????????????????????????????? ?????????? (CBD) Clostridium thermocellum, ???????????? ?????? ?????????????????? ?????????? ScFvbINF-CBD. ?????????????????? ???????????????????? ???????????????????? ?????????????????? ?????????? ?? ?????????????? Escherichia coli. ?????????????? ?????????? ???????????????? ?? ??????????????????????, ?????????????????????????? ???????????????? ?????????? ?????????? ?????????????????????? ???? ?????????????????????????? ?????????? ?????????????????? in vitro. ScFvbINF-CBD ???????????????????????????? ???? ?????????????????? ????????????????. ????????????????????. ?????????????????? CBD ?? ???????????? ?????????????????? ?????????? ???????????????????????? ?????????????????????????????? ???? ?????????????????????? ?????????????????????????? ScFvbINF-CBD ???? ???????????????????????????????????? ??????????????. ?? ?????????????? ???????????????????? ???????????????????????????? ?????? ?????????????? rhIFN-??1b ???? ?????????????? ?????????? ????????????, ???????????????????? ?????????? ?????? ?????????????????????? ???? ?????????? ??????????????????, ?? ???????????????? ?????????? 89 %. ??????????????. ?????????????????? ???????????????????????????? ???????????????????????????????????? ?????????????? ?????????????????? ???????????????? ?????????????????????????? IFN-??1b ???????????????? ???? ?????????????? ???????????????? ????????????.<br/>????????. ?????????????????? ???????????????????????? ?????????? ?????????????????? ?????????????????????????? ?????????????????????????????????? ???????????????? ?????? ?????????????????? ???? ???????????????? ?????????????????????????????? IFN-??1b ????????????. ????????????. ?????????????????????????????????? ?????????????????????????? ??????, ?????? ?????????????? ScFv ???????????????????? ???? hIFN-??1b, ???? ??????????????????????????????????????????? ?????????? (CBD) Clostri??di??um thermocellum, ???????????????? ?????? ?????????????????? ???????????????? ScFvbINF-CBD. ?????????????????? ?????????????????? ???????????????????? ?????????????????? ???????????????? ?? ???????????????? Escherichia coli. ???????????????? ?????????? ???????????????????? ?? ??????????????????, ?????????????????????????? ???????????????? ?????????? ???????????? ?????????????????????? ?? ?????????????????????????? ???????????? ?????????????????? in vitro. ScFvbINF-CBD ???????????????????????????? ???? ???????????????????? ??????????. ????????????????????. ?????????????????? CBD ???? ???????????? ?????????????????? ?????????? ???????????????????? ??????????????????????, ???? ???????????????????? ?????????????????????????? ScFvbINF-CBD ???? ???????????????????????????????? ??????????????. ???? ?????????????????? ???????????????????? ?????????????????????????? ???????????????? ???????? ???????????????? rhIFN-??1b ???? ???????????????? ???????????? ????????????, ?????????????????? ?????????? ???????? ?????????????????????? ?? ???????????? ??????????????????, ?? ???????????????? ???????????? ?????? 89 %. ????????????????. ?????????????????? ???????????????????????? ???????????????????????????????? ?????????????? ???????????????? ???????????????? ???????????????????????????? IFN-??1b ???????????? ???? ???????????????? ?????????????? ????????????.<br/>