Biopolym. Cell. 2015; 31(2):115-122.
http://dx.doi.org/10.7124/bc.0008D5
Molecular and Cell Biotechnologies
Recombinant Staphylococcal protein A with cysteine residue for preparation of affinity chromatography stationary phase and immunosensor applications
1, 2Gorbatiuk O. B., 1, 3Bakhmachuk A. O., 1Dubey L. V., 1, 3Usenko M. O., 1, 2Irodov D. M., 1, 2Okunev O. V., 1Kostenko O. M., 1Rachkov A. E., 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
  3. Educational and Scientific Center "Institute of Biology",
    Taras Shevchenko National University of Kyiv
    64/13, Volodymyrska Str., Kyiv, Ukraine, 01601

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.
Keywords: antibodies, recombinant Staphylococcal protein A, protein immobilization, affinity chromatography, immunosensor, surface plasmon resonance
Full text: (PDF, in English)

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