Biopolym. Cell. 2015; 31(4):279-284.
http://dx.doi.org/10.7124/bc.0008EC
Molecular and Cell Biotechnologies
Development of the chromatographic medium for the affinity isolation of the recombinant hIFN-β1b based on immobilized single-chain antibodies
1, 2Pokholenko Ia. O., 1, 2Gorbatiuk O. B., 1, 2Okunev O. V., 1, 2Irodov D. M., 1Degtiarova M. I., 1, 2Palivoda O. G., 1, 2Kordium V. A.
  1. Institute of Molecular Biology and Genetics, NAS of Ukraine
    150, Akademika Zabolotnoho Str., Kyiv, Ukraine, 03680
  2. State Institute of Genetic and Regenerative Medicine, NAMS of Ukraine
    67, Vyshhorodska Str., Kyiv, Ukraine, 04114

Abstract

Aim. The development of a laboratory method for the production of immunoaffinity chromatography medium for the purification of the recombinant human IFN-β1b. Methods. A gene of the chimeric protein ScFvbINF-CBD was constructed using the DNA sequences encoding ScFv specific to hIFN-β1b 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-β1b 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-β1b from the complex protein mixtures.
Keywords: Interferon-β1b, single-chain antibodies, immunoaffinity purification, cellulose-binding domain.
Full text: (PDF, in English)

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