Biopolym. Cell. 2012; 28(2):141-148.
Molecular and Cell Biotechnologies
Bioaffinity sorbent based on immobilized protein A Staphylococcus aureus: development and application
1Gorbatiuk O. B., 2Tsapenko M. V., 2, 3Pavlova M. V., 2, 3Okunev O. V., 2, 3Kordium V. A.
  1. Educational and Scientific Center "Institute of Biology",
    Taras Shevchenko National University of Kyiv
    64/13, Volodymyrska Str., Kyiv, Ukraine, 01601
  2. Institute of Molecular Biology and Genetics, NAS of Ukraine
    150, Akademika Zabolotnoho Str., Kyiv, Ukraine, 03680
  3. State Institute of Genetic and Regenerative Medicine, NAMS of Ukraine
    57/3, Chervonoarmiyska Str., Kyiv, Ukraine, 03150

Abstract

Aim. The obtaining of bioaffinity sorbent based on the immobilized protein A of S. aureus (SPA) using two cellulose-binding domains (CBD), and its application for purification of antibodies. Methods. The DNA sequences encoding SPA and two CBD were genetically fused, expressed in the high-productive Escherichia coli system and the protein SPA-CBD2 was obtained in a soluble form. The SPA-CBD2 fusion protein was affinity immobilized on the microcrystalline cellulose. Results. Capacity of bioaffinity sorbent (1 mg SPA-CBD2/1 ml CC31-cellulose), dynamic capacity (3 mg mouse IgG/1 ml bioaffinity sorbent), efficiency and stability during prolonged storage were determined. The bioffinity sorbent was used for purification of antibodies. The purity of antibodies in eluted fractions was more than 95 %. The purified antibodies detected target antigens with a high sensitivity. Conclusions. The designed bioaffinity sorbent provides obtaining pure poly- and monoclonal antibodies in functionally active form and can be useful for the fractionation of mouse immunoglobulin G.
Keywords: antibodies, protein A, cellulose-binding domain, protein immobilization, affinity chromatography

References

[1] Denizli A. 2011 Purification of antibodies by affinity chromatography Hacettepe J. Biol. Chem 39, N 1:1–18.
[2] Boi C., Dimartino S., Sarti G. C. 2008 Performance of a new protein a affinity membrane for the primary recovery of antibodies Biotechnol. Prog 24, N 3:640–647.
[3] Hober S., Nord K., Linhult M. 2007 Protein A chromatography for antibody purification J. Chromatogr. B. Analyt Technol. Biomed. Life Sci 848, N 1:40–47.
[4] Hickstein H., Korten G., Bast R., Barz D., Templin R., Schneidewind J. M., Kittner C., Nizze H., Schmidt R. 1998 Protein A immunoadsorption (i. a.) in renal transplantation patients with vascular rejection Transfus. Sci 19:53–57.
[5] Moks T., Abrahmsen L., Nilsson B., Hellman U., Sjoquist J., Uhlen M. 1986 Staphylococcal protein A consists of five IgG-binding domains Eur. J. Biochem 156, N 3:637–643.
[6] Ghose S., Hubbard B., Cramer S. M. 2006 Evaluation and comparison of alternatives to Protein A chromatography mimetic and hydrophobic charge induction chromatographic stationary phases J. Chromatogr. A 1122, N 1–2:144–152.
[7] Gottschalk U. 2009 Process scale purification of antibodies Hoboken: Wiley & Sons, 430 p.
[8] Housden N. G, Harrison S., Roberts S. E., Beckingham J. A., Graille M., Stura E., Gore M. G. 2003 Immunoglobulin-binding domains: Protein L from Peptostreptococcus magnus Biochem. Soc. Trans 31, Pt 3:716–718.
[9] Affinity chromatography: methods and protocols 2000 Eds P. Bailon et al New York: Humana press, Vol. 147 230 p.
[10] Morag E., Lapidot A., Govorko D., Lamed R., Wilchek M., Bayer E. A., Shoham Y. 1995 Expression, purification, and characterization of the cellulose-binding domain of the scaffoldin subunit from the cellulosome of Clostridium thermocellum Appl. Environ. Microbiol 61, N 5:1980–1986.
[11] Gilchuk P. V., Volkov G. L. 2006 Immobilization of mouse singlechain antibodies for affinity chromatography using the cellulose-binding protein Ukr. Biokhim. Zh. 78, N 4 P. 160–163.
[12] Studier F. W. 2005 Protein production by auto-induction in high density shaking cultures Protein Expr. Purif 41, N 1 P. 207–234.
[13] Westermeir R. 1997 Electrophoresis in practice: a guide to methods and application of DNA and protein separations Weinheim: VCH, 331 p.
[14] Ljungquist C., Jansson B., Moks T., Uhleen M. 1989 Thiol-directed immobilization of recombinant IgG-binding receptors Eur. J. Biochem 186, N 3:557–561.
[15] Linhult M., Gulich S., Graslund T., Nygren P. A., Hober S. 2003 Evaluation of different linker regions for multimerization and coupling chemistry for immobilization of a proteinaceous affinity ligand Protein Eng 16, N 12:1147–1152.
[16] Atkins K. L., Burman J. D., Chamberlain E. S., Cooper J. E., Poutrel B., Bagby S., Jenkins A. T., Feil E. J., van den Elsen J. M. 2008 S. aureus IgG-binding proteins SpA and Sbi: host specificity and mechanisms of immune complex formation Mol. Immunol 45, N 6:1600–1611.
[17] Tormo J., Lamed R., Chirino A. J., Morag E., Bayer E. A., Shoham Y., Steitz T. A. 1996 Crystal structure of a bacterial family-III cellulose-binding domain: a general mechanism for attachment to cellulose EMBO J 15, N 21:5739–5751.
[18] Lidner M., Salovuori I., Ruohonen L., Teeri T. T. 1996 Characterization of a double cellulose-binding domain: synergistic high-affinity binding to crystalline cellulose J. Biol. Chem 271, N 35:21268–21272.
[19] Pat. USA N 5837814. 1998. Cellulose binding domain proteins O. Shoseyov, K. Yosef, I. Shpiegl, M. Goldstein, R. Doi.
[20] Arakawa T., Philo J. S., Tsumoto K., Yumioka R., Ejimac D. 2004 Elution of antibodies from a Protein-A column by aqueous arginine solutions Protein Expr. Purif 36, N 2:244–248.
[21] Huang B., Liu F. F., Dong X. Y., Sun Y. 2011 Molecular mechanism of the affinity interactions between protein A and human immunoglobulin G1 revealed by molecular aimulations J. Phys. Chem. B 115, N 14:4168–4176.