Skip to main content
SpringerLink
Log in

Immobilization of Bioactive Protein A from Staphylococcus aureus (SpA) on the Surface of Bacillus subtilis Spores

  • Research
  • Published:
Molecular Biotechnology Aims and scope Submit manuscript

Abstract

Protein A from Staphylococcus aureus (SpA) is a 40–60 kDa cell-wall component, composed of five homologous immunoglobulin (Ig)-binding domains folded into a three-helix bundle. Each of these five domains is able to bind Igs from many different mammalian species. Recombinant SpA is widely used as a component of diagnostic kits for the detection and purification of IgGs from serum or other biological fluids. In this study, purified SpA was adsorbed and covalently linked to Bacillus subtilis spores. Spores are extremely stable cell forms and are considered as an attractive platform to display heterologous proteins. A sample containing about 36 μg of SpA was covalently immobilized on the surface of 4 × 1010 spores. Spore-bound SpA retained its IgG-binding activity, even after seven consecutive binding and washing steps, suggesting that it can be recycled and utilized several times. FACS analysis revealed that spores with covalently attached SpA had significantly improved fluorescence intensities when compared to those of spores with adsorbed SpA, suggesting that the covalent approach is more efficient than sole adsorption regarding protein attachment to the spore surface.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Fig. 1
Fig. 2
Fig. 3
Fig. 4
Fig. 5
Fig. 6
Fig. 7
Fig. 8
Fig. 9

Similar content being viewed by others

References

  1. Wu, C. H., Mulchandani, A., & Chen, W. (2008). Versatile microbial surface-display for environmental remediation and biofuels production. Trends in Microbiology, 16, 181–188.

    Article  CAS  Google Scholar 

  2. Lee, S. Y., Choi, J. H., & Xu, Z. (2003). Microbial cell-surface display. Trends in Biotechnology, 21, 45–52.

    Article  CAS  Google Scholar 

  3. Cutting, S. M., Hong, H. A., Baccigalupi, L., & Ricca, E. (2009). Oral vaccine delivery by recombinant spore probiotics. International Reviews of Immunology, 28, 487–505.

    Article  CAS  Google Scholar 

  4. Du, C., Chan, W. C., McKeithan, T. W., & Nickerson, K. W. (2005). Surface display of recombinant proteins on Bacillus thuringiensis spores. Applied and Environment Microbiology, 71, 3337–3341.

    Article  CAS  Google Scholar 

  5. Isticato, R., & Ricca, E. (2014). Spore surface display. Microbiology Spectrum. doi:10.1128/microbiolspec.TBS-0011-2012.

    Google Scholar 

  6. Henriques, A. O., & Moran, C. P, Jr. (2007). Structure, assembly, and function of the spore surface layers. Annual Review of Microbiology, 61, 555–588.

    Article  CAS  Google Scholar 

  7. McKenney, P. T., Driks, A., Eskandarian, H. A., Grabowski, P., Guberman, J., Wang, K. H., et al. (2010). A distance-weighted interaction map reveals a previously uncharacterized layer of the Bacillus subtilis spore coat. Current Biology, 20, 934–938.

    Article  CAS  Google Scholar 

  8. Imamura, D., Kuwana, R., Takamatsu, H., & Watabe, K. (2011). Proteins involved in formation of the outermost layer of Bacillus subtilis spores. Journal of Bacteriology, 193, 4075–4080.

    Article  CAS  Google Scholar 

  9. Ricca, E., Baccigalupi, L., Cangiano, G., De Felice, M., & Isticato, R. (2014). Mucosal vaccine delivery by non-recombinant spores of Bacillus subtilis. Microbial Cell Factories, 13, 115.

    Google Scholar 

  10. Starovasnik, M. A., O’Connell, M. P., Fairbrother, W. J., & Kelley, R. F. (1999). Antibody variable region binding by Staphylococcal protein A: Thermodynamic analysis and location of the Fv binding site on E-domain. Protein Science, 8, 1423–1431.

    Article  CAS  Google Scholar 

  11. Lu, H. C., Chen, H. M., Lin, Y. S., & Lin, J. W. (2000). A reusable and specific protein A-coated piezoelectric biosensor for flow injection immunoassay. Biotechnology Progress, 16, 116–124.

    Article  CAS  Google Scholar 

  12. Anderson, G. P., Jacoby, M. A., Ligler, F. S., & King, K. D. (1997). Effectiveness of protein A for antibody immobilization for a fiber optic biosensor. Biosensors and Bioelectronics, 12, 329–336.

    Article  CAS  Google Scholar 

  13. Gorbatiuk, O. B., Tsapenko, M. V., Pavlova, M. V., Okunev, O. V., & Kordium, V. A. (2012). Bioaffinity sorbent based on immobilized protein A Staphylococcus aureus: Development and application. Biopolymers and Cell, 28, 141–148.

    Article  CAS  Google Scholar 

  14. Ghose, S., Allen, M., Hubbard, B., Brooks, C., & Cramer, S. M. (2005). Antibody variable region interactions with protein A: Implications for the development of generic purification processes. Biotechnology and Bioengineering, 92, 665–673.

    Article  CAS  Google Scholar 

  15. Widjojoatmodjo, M. N., Fluit, A. C., Torensma, R., & Verhoef, J. (1993). Comparison of immunomagnetic beads coated with protein A, protein G, or goat anti-mouse immunoglobulins. Applications in enzyme immunoassays and immunomagnetic separations. Journal of Immunological Methods, 165, 11–19.

    Article  CAS  Google Scholar 

  16. Owaku, K., Goto, M., Ikariyama, Y., & Aizawa, M. (1995). Protein A Langmuir-Blodgett film for antibody immobilization and its use in optical immunosensing. Analytical Chemistry, 67, 1613–1616.

    Article  CAS  Google Scholar 

  17. Cao, Y., Tian, W., Gao, S., Yu, Y., Yang, W., & Bai, G. (2007). Immobilization staphylococcal protein a on magnetic cellulose microspheres for IgG affinity purification. Artificial Cells, Blood Substitutes, and Immobilization Biotechnology, 35, 467–480.

    Article  CAS  Google Scholar 

  18. Sambrook, J., & Russell, D. W. (2001). Molecular cloning: A laboratory manual (Vol. 3). Cold Spring Harbor: Cold Spring Harbor Laboratory Press.

    Google Scholar 

  19. QIAGEN. (2001). NI-NTA magnetic agarose beads handbook. For manual and automated assays using 6xHis-tagged proteins purification of 6xHis-tagged proteins (2nd ed.). QIAGEN.

  20. Dedonder, R. A., Lepesant, J. A., Lepesant-Kejzlarova, J., Billault, A., Steinmetz, M., & Kunst, F. (1977). Construction of a kit of reference strains for rapid genetic mapping in Bacillus subtilis 168. Applied and Environment Microbiology, 33, 989–993.

    CAS  Google Scholar 

  21. Nicholson, W. L., & Setlow, P. (1990). Molecular biological methods for Bacillus (pp. 391–450)., Sporulation, germination and outgrowth Chichester: Wiley.

    Google Scholar 

  22. Huang, J. M., Hong, H. A., Van Tong, H., Hoang, T. H., Brisson, A., & Cutting, S. M. (2010). Mucosal delivery of antigens using adsorption to bacterial spores. Vaccine, 28, 1021–1030.

    Article  CAS  Google Scholar 

  23. Kyte, J., & Doolittle, R. F. (1982). A simple method for displaying the hydropathic character of a protein. Journal of Molecular Biology, 157, 105–132.

    Article  CAS  Google Scholar 

  24. Carroll, A. M., Plomp, M., Malkin, A. J., & Setlow, P. (2008). Protozoal digestion of coat-defective Bacillus subtilis spores produces “rinds” composed of insoluble coat protein. Applied and Environment Microbiology, 74, 5875–5881.

    Article  CAS  Google Scholar 

  25. Singh, R. K., Zhang, Y. W., Nguyen, N. P., Jeya, M., & Lee, J. K. (2011). Covalent immobilization of beta-1,4-glucosidase from Agaricus arvensis onto functionalized silicon oxide nanoparticles. Applied Microbiology and Biotechnology, 89, 337–344.

    Article  CAS  Google Scholar 

  26. Hobbs, H., Reddy, D., Rajeshwari, R., & Reddy, A. (1987). Use of direct antigen coating and protein A coating ELISA procedures for detection of three peanut viruses. Plant Disease, 71, 747–749.

    Article  Google Scholar 

  27. Gashtasbi, F., Ahmadian, G., & Noghabi, K. A. (2014). New insights into the effectiveness of alpha-amylase enzyme presentation on the Bacillus subtilis spore surface by adsorption and covalent immobilization. Enyzme and Microbial Technology, 64–65, 17–23.

    Article  Google Scholar 

  28. Barbosa, O., Torres, R., Ortiz, C., Berenguer-Murcia, A., Rodrigues, R. C., & Fernandez-Lafuente, R. (2013). Heterofunctional supports in enzyme immobilization: From traditional immobilization protocols to opportunities in tuning enzyme properties. Biomacromolecules, 14, 2433–2462.

    Article  CAS  Google Scholar 

  29. Pessela, B. C., Mateo, C., Fuentes, M., Vian, A., Garcia, J. L., Carrascosa, A. V., et al. (2004). Stabilization of a multimeric beta-galactosidase from Thermus sp. strain T2 by immobilization on novel heterofunctional epoxy supports plus aldehyde-dextran cross-linking. Biotechnology Progress, 20, 388–392.

    Article  CAS  Google Scholar 

  30. Graille, M., Stura, E. A., Corper, A. L., Sutton, B. J., Taussig, M. J., Charbonnier, J. B., & Silverman, G. J. (2000). Crystal structure of a Staphylococcus aureus protein A domain complexed with the Fab fragment of a human IgM antibody: Structural basis for recognition of B-cell receptors and superantigen activity. Proceedings of the National Academy of Sciences of the United States of America, 97, 5399–5404.

    Article  CAS  Google Scholar 

  31. Gashtasbi, F., Ahmadian, G., & Noghabi, K. A. (2014). New insights into the effectiveness of alpha-amylase enzyme presentation on the Bacillus subtilis spore surface by adsorption and covalent immobilization. Enzyme and Microbial Technology, 64–65, 17–23.

    Article  Google Scholar 

  32. Cho, E. A., Kim, E. J., & Pan, J. G. (2011). Adsorption immobilization of Escherichia coli phytase on probiotic Bacillus polyfermenticus spores. Enyzme and Microbial Technology, 49, 66–71.

    Article  CAS  Google Scholar 

  33. le Duc, H., Hong, H. A., Fairweather, N., Ricca, E., & Cutting, S. M. (2003). Bacterial spores as vaccine vehicles. Infection and Immunity, 71, 2810–2818.

    Article  CAS  Google Scholar 

  34. Hoang, T. H., Hong, H. A., Clark, G. C., Titball, R. W., & Cutting, S. M. (2008). Recombinant Bacillus subtilis expressing the Clostridium perfringens alpha toxoid is a candidate orally delivered vaccine against necrotic enteritis. Infection and Immunity, 76, 5257–5265.

    Article  CAS  Google Scholar 

  35. Huang, J. M., La Ragione, R. M., Cooley, W. A., Todryk, S., & Cutting, S. M. (2008). Cytoplasmic delivery of antigens, by Bacillus subtilis enhances Th1 responses. Vaccine, 26, 6043–6052.

    Article  CAS  Google Scholar 

  36. Yim, S. K., Jung, H. C., Yun, C. H., & Pan, J. G. (2009). Functional expression in Bacillus subtilis of mammalian NADPH-cytochrome P450 oxidoreductase and its spore-display. Protein Expression and Purification, 63, 5–11.

    Article  CAS  Google Scholar 

  37. Ghose, S., Hubbard, B., & Cramer, S. M. (2007). Binding capacity differences for antibodies and Fc-fusion proteins on protein A chromatographic materials. Biotechnology and Bioengineering, 96, 768–779.

    Article  CAS  Google Scholar 

  38. Steidler, L., Remaut, E., & Fiers, W. (1993). Pap pili as a vector system for surface exposition of an immunoglobulin G-binding domain of protein A of Staphylococcus aureus in Escherichia coli. Journal of Bacteriology, 175, 7639–7643.

    CAS  Google Scholar 

  39. Samuelson, P., Gunneriusson, E., Nygren, P. A., & Stahl, S. (2002). Display of proteins on bacteria. Journal of Biotechnology, 96, 129–154.

    Article  CAS  Google Scholar 

Download references

Acknowledgments

The authors would like to thank the National Institute of Genetic Engineering and Biotechnology (NIGEB) of Iran for providing the necessary equipments and facilities. Funding for the project was provided by the Iran National Science Foundation (Project No. 90004656).

Author information

Authors and Affiliations

  1. Department of Industrial and Environmental Biotechnology, National Institute of Genetic Engineering and Biotechnology (NIGEB), Tehran, Iran

    Samira Ghaedmohammadi, Garshasb Rigi & Gholamreza Ahmadian

  2. Chemistry and Chemical Engineering Research Center of Iran, Tehran, Iran

    Reza Zadmard

  3. Department of Structural and Functional Biology, Federico II University of Naples, Naples, Italy

    Ezio Ricca

Authors
  1. Samira Ghaedmohammadi
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Garshasb Rigi
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Reza Zadmard
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Ezio Ricca
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Gholamreza Ahmadian
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Gholamreza Ahmadian.

Rights and permissions

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Ghaedmohammadi, S., Rigi, G., Zadmard, R. et al. Immobilization of Bioactive Protein A from Staphylococcus aureus (SpA) on the Surface of Bacillus subtilis Spores. Mol Biotechnol 57, 756–766 (2015). https://doi.org/10.1007/s12033-015-9868-z

Download citation

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s12033-015-9868-z

Keywords

Search

Navigation