Biopolym. Cell. 2015; 31(2):115-122.
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


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


[1] Sidorin EV, Solov'eva TF. IgG-binding proteins of bacteria. Biochemistry (Mosc). 2011;76(3):295-308.
[2] Forsgren A, Sj?quist J. "Protein A" from S. aureus. I. Pseudo-immune reaction with human gamma-globulin. J Immunol. 1966;97(6):822-7.
[3] Abrahms?n L, Moks T, Nilsson B, Hellman U, Uhl?n M. Analysis of signals for secretion in the staphylococcal protein A gene. EMBO J. 1985;4(13B):3901-6.
[4] Moks T, Abrahms?n L, Nilsson B, Hellman U, Sj?quist J, Uhl?n M. Staphylococcal protein A consists of five IgG-binding domains. Eur J Biochem. 1986;156(3):637-43.
[5] Sjodahl J. Repetitive sequences in protein A from Staphylococcus aureus. Arrangement of five regions within the protein, four being highly homologous and Fc-binding. Eur J Biochem. 1977;73(2):343-51.
[6] Hober S, Nord K, Linhult M. Protein A chromatography for antibody purification. J Chromatogr B Analyt Technol Biomed Life Sci. 2007;848(1):40-7.
[7] Makaraviciute A, Ramanaviciene A. Site-directed antibody immobilization techniques for immunosensors. Biosens Bioelectron. 2013;50:460-71.
[8] Rachkov A, Holodova Y, Ushenin Y, Miroshnichenko D, Te­legeev G, Soldatkin A. Development of bioselective element of SPR spectrometer for monitoring of oligonucleotide interactions and comparison with thermodynamic calculati­ons. Sens Lett. 2009;7(5):957–61.
[9] Kanno S, Yanagida Y, Haruyama T, Kobatake E, Aizawa M. Assembling of engineered IgG-binding protein on gold surface for highly oriented antibody immobilization. J Biotechnol. 2000;76(2-3):207-14.
[10] Dubois L, Nuzzo R. Synthesis, structure, and properties of mo­del organic surfaces. Annu Rev Phys Chem. 1992; 43(1): 437–63.
[11] Wong LS, Khan F, Micklefield J. Selective covalent protein immobilization: strategies and applications. Chem Rev. 2009;109(9):4025-53.
[12] Hermanson GT. Bioconjugate techniques. 2nd Ed. Ams­ter­dam: «Academic Press», 2008. 1202p.
[13] Venkatesan N, Kim BH. Peptide conjugates of oligonucleotides: synthesis and applications. Chem Rev. 2006;106(9):3712-61.
[14] Lu K, Duan QP, Ma L, Zhao DX. Chemical strategies for the synthesis of peptide-oligonucleotide conjugates. Bioconjug Chem. 2010;21(2):187-202.
[15] Peng H, Chen W, Cheng Y, Hakuna L, Strongin R, Wang B. Thiol reactive probes and chemosensors. Sensors (Basel). 2012;12(11):15907-46.
[16] Krchn?k V, V?gner J, Safar P, Lebl M. Noninvasive continuous monitoring of solid-phase peptide synthesis by acid-ba­se indicator. Collect Czech Chem Commun. 1988; 53(11): 2542–48.
[17] Keller O, Rudinger J. Preparation and some properties of maleimido acids and maleoyl derivatives of peptides. Helv Chim Acta. 1975;58(2):531-41.
[18] Song HY, Ngai MH, Song ZY, MacAry PA, Hobley J, Lear MJ. Practical synthesis of maleimides and coumarin-linked probes for protein and antibody labelling via reduction of native disulfides. Org Biomol Chem. 2009;7(17):3400-6.
[19] Studier FW. Protein production by auto-induction in high density shaking cultures. Protein Expr Purif. 2005;41(1):207-34.
[20] Westermeir R. Electrophoresis in practice: a guide to me­thods and application of DNA and protein separations. Wei­nheim: «VCH», 1997. 331 p.
[21] Oligonucleotide synthesis: a practical approach. Ed. Gait MJ. Oxford: «IRL Press», 1984. 217 p.
[22] Mallik R, Wa C, Hage DS. Development of sulfhydryl-reactive silica for protein immobilization in high-performance affinity chromatography. Anal Chem. 2007;79(4):1411-24.