Recombinant Staphylococcal protein A with cysteine residue for preparation of affinity chromatography stationary phase and immunosensor applications

Authors

  • O. B. Gorbatiuk Institute of Molecular Biology and Genetics, NAS of Ukraine 150, Akademika Zabolotnoho Str., Kyiv, Ukraine, 03680; State Institute of Genetic and Regenerative Medicine, NAMS of Ukraine 67, Vyshhorodska Str., Kyiv, Ukraine, 04114 Author
  • A. O. Bakhmachuk Institute of Molecular Biology and Genetics, NAS of Ukraine 150, Akademika Zabolotnoho Str., Kyiv, Ukraine, 03680; Educational and Scientific Center "Institute of Biology", Taras Shevchenko National University of Kyiv 64/13, Volodymyrska Str., Kyiv, Ukraine, 01601 Author
  • L. V. Dubey Institute of Molecular Biology and Genetics, NAS of Ukraine 150, Akademika Zabolotnoho Str., Kyiv, Ukraine, 03680 Author
  • M. O. Usenko Institute of Molecular Biology and Genetics, NAS of Ukraine 150, Akademika Zabolotnoho Str., Kyiv, Ukraine, 03680; Educational and Scientific Center "Institute of Biology", Taras Shevchenko National University of Kyiv 64/13, Volodymyrska Str., Kyiv, Ukraine, 01601 Author
  • D. M. Irodov Institute of Molecular Biology and Genetics, NAS of Ukraine 150, Akademika Zabolotnoho Str., Kyiv, Ukraine, 03680; State Institute of Genetic and Regenerative Medicine, NAMS of Ukraine 67, Vyshhorodska Str., Kyiv, Ukraine, 04114 Author
  • O. V. Okunev Institute of Molecular Biology and Genetics, NAS of Ukraine 150, Akademika Zabolotnoho Str., Kyiv, Ukraine, 03680; State Institute of Genetic and Regenerative Medicine, NAMS of Ukraine 67, Vyshhorodska Str., Kyiv, Ukraine, 04114 Author
  • O. M. Kostenko Institute of Molecular Biology and Genetics, NAS of Ukraine 150, Akademika Zabolotnoho Str., Kyiv, Ukraine, 03680 Author
  • A. E. Rachkov Institute of Molecular Biology and Genetics, NAS of Ukraine 150, Akademika Zabolotnoho Str., Kyiv, Ukraine, 03680 Author
  • V. A. Kordium Institute of Molecular Biology and Genetics, NAS of Ukraine 150, Akademika Zabolotnoho Str., Kyiv, Ukraine, 03680; State Institute of Genetic and Regenerative Medicine, NAMS of Ukraine 67, Vyshhorodska Str., Kyiv, Ukraine, 04114 Author

DOI:

https://doi.org/10.7124/bc.0008D5

Keywords:

antibodies, recombinant Staphylococcal protein A, protein immobilization, affinity chromatography, immunosensor, surface plasmon resonance

Abstract

Aim. Engineering of recombinant Staphylococcal protein A with cysteine residue (SPA-Cys) for preparation of affinity chromatography stationary phase and formation of bioselective element of immunosensor. Methods. DNA sequences encoding IgG-binding region of SPA, His-tag and cysteine were genetically fused and expressed in E. coli. SPA-Cys was immobilized on maleimide-functionalized silica beads for affinity chromatography stationary phase preparation and on a gold sensor surface as a bioselective element of immunosensor. Results. SPA-Cys was expressed at a high-level in a soluble form. The target protein was purified and showed a high IgG-binding activity. The capacity of the obtained SPA-Cys-based affinity chromatography stationary phase was 10–12 mg of IgG /ml. The purity of eluted IgG was more than 95 % in one-step purification procedure. The developed SPA-Cys-based bioselective element of immunosensor selectively interacted with human IgG and did not interact with the control proteins. Conclusions. The recombinant Staphylococcal protein A with cysteine residue was successfully used for the preparation of affinity chromatography stationary phase and formation of the bioselective element of immunosensor.

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Published

2015-04-20

Issue

Vol. 31 No. 2 (2015)

Section

Molecular and Cell Biotechnologies