Molecular Biotechnology

, Volume 60, Issue 3, pp 215–225 | Cite as

The Optimisation of the Expression of Recombinant Surface Immunogenic Protein of Group B Streptococcus in Escherichia coli by Response Surface Methodology Improves Humoral Immunity

  • Diego A. Díaz-Dinamarca
  • José I. Jerias
  • Daniel A. Soto
  • Jorge A. Soto
  • Natalia V. Díaz
  • Yessica Y. Leyton
  • Rodrigo A. Villegas
  • Alexis M. Kalergis
  • Abel E. VásquezEmail author
Original Paper


Group B Streptococcus (GBS) is the leading cause of neonatal meningitis and a common pathogen in livestock and aquaculture industries around the world. Conjugate polysaccharide and protein-based vaccines are under development. The surface immunogenic protein (SIP) is a conserved protein in all GBS serotypes and has been shown to be a good target for vaccine development. The expression of recombinant proteins in Escherichia coli cells has been shown to be useful in the development of vaccines, and the protein purification is a factor affecting their immunogenicity. The response surface methodology (RSM) and Box–Behnken design can optimise the performance in the expression of recombinant proteins. However, the biological effect in mice immunised with an immunogenic protein that is optimised by RSM and purified by low-affinity chromatography is unknown. In this study, we used RSM for the optimisation of the expression of the rSIP, and we evaluated the SIP-specific humoral response and the property to decrease the GBS colonisation in the vaginal tract in female mice. It was observed by NI–NTA chromatography that the RSM increases the yield in the expression of rSIP, generating a better purification process. This improvement in rSIP purification suggests a better induction of IgG anti-SIP immune response and a positive effect in the decreased GBS intravaginal colonisation. The RSM applied to optimise the expression of recombinant proteins with immunogenic capacity is an interesting alternative in the evaluation of vaccines in preclinical phase, which could improve their immune response.


Group B streptococcus Response surface methodology Protein expression 



The authors would like to thank Miguel Muñoz, Selene Espinosa, Magaly Barrientos and América Abarca for their technical assistance and Luis Vidal for editing the English text.

Authors’ Contributions

JJ, JS and DD-D performed all experimental procedures, data collection, analysis, interpretation and the writing of this paper. JS, DD-D and DS developed the experimental rSIP purification procedures and the ELISA procedure. RV performed the statistical analysis and interpretation. AMK and AEV analysed and interpreted the results. AEV designed and directed the experiments and wrote and edited this paper. All authors read and approved the final manuscript.


This research was supported by the Instituto de Salud Pública de Chile and Millenium Institute on Immunology and Immunotherapy.

Compliance with Ethical Standards

Conflict of interest

The authors declare that they have no conflict of interest.

Supplementary material

12033_2018_65_MOESM1_ESM.docx (13 kb)
Supplementary material 1 (DOCX 13 kb)


  1. 1.
    Rato, M. G., Bexiga, R., Florindo, C., Cavaco, L. M., Vilela, C. L., & Santos-Sanches, I. (2013). Antimicrobial resistance and molecular epidemiology of streptococci from bovine mastitis. Veterinary Microbiology, 161(3), 286–294. Scholar
  2. 2.
    Mahmmod, Y. S., Klaas, I. C., Katholm, J., Lutton, M., & Zadoks, R. N. (2015). Molecular epidemiology and strain-specific characteristics of Streptococcus agalactiae at the herd and cow level. Journal of Dairy Science, 98(10), 6913–6924. Scholar
  3. 3.
    Pinto, T. C. A., Costa, N. S., Corrêa, A. B. D. A., Oliveira, I. C. M. D., Mattos, M. C. D., Rosado, A. S., et al. (2014). Conjugative transfer of resistance determinants among human and bovine Streptococcus agalactiae. Brazilian Journal of Microbiology, 45(3), 785–789. Scholar
  4. 4.
    Li, W., Su, Y. L., Mai, Y. Z., Li, Y. W., Mo, Z. Q., & Li, A. X. (2014). Comparative proteome analysis of two Streptococcus agalactiae strains from cultured tilapia with different virulence. Veterinary Microbiology, 170(1), 135–143. Scholar
  5. 5.
    Bekker, V., Bijlsma, M. W., van de Beek, D., Kuijpers, T. W., & van der Ende, A. (2014). Incidence of invasive group B streptococcal disease and pathogen genotype distribution in newborn babies in the Netherlands over 25 years: A nationwide surveillance study. The Lancet Infectious Diseases, 14(11), 1083–1089. Scholar
  6. 6.
    Le Doare, K., & Heath, P. T. (2013). An overview of global GBS epidemiology. Vaccine, 31, D7–D12. Scholar
  7. 7.
    Heath, P. T. (2016). Status of vaccine research and development of vaccines for GBS. Vaccine. Scholar
  8. 8.
    Verani, J. R., McGee, L., & Schrag, S. J. (2010). Prevention of perinatal group B streptococcal disease: Revised guidelines from CDC, 2010. Atlanda: Department of Health and Human Services, Centres for Disease Control and Prevention.Google Scholar
  9. 9.
    Emaneini, M., Jabalameli, F., Mirsalehian, A., Ghasemi, A., & Beigverdi, R. (2016). Characterisation of virulence factors, antimicrobial resistance pattern and clonal complexes of group B streptococci isolated from neonates. Microbial Pathogenesis, 99, 119–122.CrossRefGoogle Scholar
  10. 10.
    Leroux-Roels, G., Maes, C., Willekens, J., De Boever, F., de Rooij, R., Martell, L., et al. (2016). A randomized, observer-blind Phase Ib study to identify formulations and vaccine schedules of a trivalent Group B Streptococcus vaccine for use in non-pregnant and pregnant women. Vaccine, 34(15), 1786–1791. Scholar
  11. 11.
    Beigverdi, R., Jabalameli, F., Mirsalehian, A., Hantoushzadeh, S., Boroumandi, S., Taherikalani, M., et al. (2014). Virulence factors, antimicrobial susceptibility and molecular characterization of Streptococcus agalactiae isolated from pregnant women. Acta Microbiologica et Immunologica Hungarica, 61(4), 425–434. Scholar
  12. 12.
    He, Y., Wang, K. Y., Xiao, D., Chen, D. F., Huang, L., Liu, T., et al. (2014). A recombinant truncated surface immunogenic protein (tSip) plus adjuvant FIA confers active protection against Group B streptococcus infection in tilapia. Vaccine, 32(51), 7025–7032. Scholar
  13. 13.
    Xu, H., Hu, C., Gong, R., Chen, Y., Ren, N., Xiao, G., et al. (2011). Evaluation of a novel chimeric B cell epitope-based vaccine against mastitis induced by either Streptococcus agalactiae or Staphylococcus aureus in mice. Clinical and Vaccine Immunology, 18(6), 893–900. Scholar
  14. 14.
    Xue, G., Yu, L., Li, S., & Shen, X. (2010). Intranasal immunization with GBS surface protein Sip and ScpB induces specific mucosal and systemic immune responses in mice. FEMS Immunology and Medical Microbiology, 58(2), 202–210. Scholar
  15. 15.
    Brodeur, B. R., Boyer, M., Charlebois, I., Hamel, J., Couture, F., Rioux, C. R., et al. (2000). Identification of group B streptococcal Sip protein, which elicits cross-protective immunity. Infection and Immunity, 68(10), 5610–5618.CrossRefGoogle Scholar
  16. 16.
    Zhang, L., Zeng, Z., Hu, C., Bellis, S. L., Yang, W., Su, Y., et al. (2016). Controlled and targeted release of antigens by intelligent shell for improving applicability of oral vaccines. Biomaterials, 77, 307–319.CrossRefGoogle Scholar
  17. 17.
    Nascimento, I. P., & Leite, L. C. C. (2012). Recombinant vaccines and the development of new vaccine strategies. Brazilian Journal of Medical and Biological Research, 45(12), 1102–1111. Scholar
  18. 18.
    Mandenius, C. F., & Brundin, A. (2008). Bioprocess optimization using design of experiments methodology. Biotechnology Progress, 24(6), 1191–1203. Scholar
  19. 19.
    Ashengroph, M., Nahvi, I., & Amini, J. (2013). Application of Taguchi design and response surface methodology for improving conversion of isoeugenol into vanillin by resting cells of Psychrobacter sp. CSW4. Iranian Journal of Pharmaceutical Research, 12(3), 411–421.Google Scholar
  20. 20.
    Strohalm, M., Kavan, D., Novak, P., Volny, M., & Havlicek, V. (2010). mMass 3: A cross-platform software environment for precise analysis of mass spectrometric data. Analytical Chemistry, 82(11), 4648–4651. Scholar
  21. 21.
    De León, A., Jiménez-Islas, H., González-Cuevas, M., & de la Rosa, A. P. B. (2004). Analysis of the expression of the Trichoderma harzianum ech42 gene in two isogenic clones of Escherichia coli by surface response methodology. Process Biochemistry, 39(12), 2173–2178.CrossRefGoogle Scholar
  22. 22.
    STATGRAPHICS Centurion XVI (Version 16.1.11). (2010). StatPoint Technologies, Inc., Herndon, VA.Google Scholar
  23. 23.
    R Core Team. (2014). R: A language and environment for statistical computing. Vienna: R Foundation for Statistical Computing. Accessed 9 Feb 2018.
  24. 24.
    Randis, T. M., Gelber, S. E., Hooven, T. A., Abellar, R. G., Akabas, L. H., Lewis, E. L., et al. (2014). Group B Streptococcus β-hemolysin/cytolysin breaches maternal-fetal barriers to cause preterm birth and intrauterine fetal demise in vivo. The Journal of Infectious Diseases, 210(2), 265–273.CrossRefGoogle Scholar
  25. 25.
    Rioux, S., Martin, D., Ackermann, H. W., Dumont, J., Hamel, J., & Brodeur, B. R. (2001). Localisation of surface immunogenic protein on group B streptococcus. Infection and Immunity, 69(8), 5162–5165. Scholar
  26. 26.
    Martin, D., Rioux, S., Gagnon, E., Boyer, M., Hamel, J., Charland, N., et al. (2002). Protection from group B streptococcal infection in neonatal mice by maternal immunisation with recombinant Sip protein. Infection and Immunity, 70(9), 4897–4901. Scholar
  27. 27.
    Scheiblhofer, S., Laimer, J., Machado, Y., Weiss, R., & Thalhamer, J. (2017). Influence of protein fold stability on immunogenicity and its implications for vaccine design. Expert Review of Vaccines, 16(5), 479–489.CrossRefGoogle Scholar
  28. 28.
    Musacchio, A., Carmenate, T., Delgado, M., & González, S. (1997). Recombinant Opc meningococcal protein, folded in vitro, elicits bactericidal antibodies after immunisation. Vaccine, 15(6–7), 751–758.CrossRefGoogle Scholar
  29. 29.
    Bolanos-Garcia, V. M., & Davies, O. R. (2006). Structural analysis and classification of native proteins from E. coli commonly co-purified by immobilised metal affinity chromatography. Biochimica et Biophysica Acta BBA-General Subjects, 1760(9), 1304–1313.CrossRefGoogle Scholar
  30. 30.
    Khan, M. A., Hamid, R., Ahmad, M., Abdin, M. Z., & Javed, S. (2010). Optimisation of culture media for enhanced chitinase production from a novel strain of Stenotrophomonas maltophilia using response surface methodology. Journal of Microbiology and Biotechnology, 20(11), 1597–1602.CrossRefGoogle Scholar
  31. 31.
    Papaneophytou, C. P., & Kontopidis, G. (2014). Statistical approaches to maximise recombinant protein expression in Escherichia coli: A general review. Protein Expression and Purification, 94, 22–32. Scholar
  32. 32.
    Einsfeldt, K., Júnior, J. B. S., Argondizzo, A. P. C., Medeiros, M. A., Alves, T. L. M., Almeida, R. V., et al. (2011). Cloning and expression of protease ClpP from Streptococcus pneumoniae in Escherichia coli: Study of the influence of kanamycin and IPTG concentration on cell growth, recombinant protein production and plasmid stability. Vaccine, 29(41), 7136–7143. Scholar
  33. 33.
    Jafari, R., Sundström, B. E., & Holm, P. (2011). Optimisation of production of the anti-keratin 8 single-chain Fv TS1-218 in Pichia pastoris using design of experiments. Microbial Cell Factories, 10(1), 34. Scholar
  34. 34.
    Larentis, A. L., Sampaio, H. D. C. C., Martins, O. B., Rodrigues, M. I., & Alves, T. L. M. (2011). Influence of induction conditions on the expression of carbazole dioxygenase components (CarAa, CarAc, and CarAd) from Pseudomonas stutzeri in recombinant Escherichia coli using experimental design. Journal of Industrial Microbiology and Biotechnology, 38(8), 1045–1054. Scholar
  35. 35.
    Maldonado, L. M. P., Hernández, V. E. B., Rivero, E. M., de la Rosa, A. P. B., Flores, J. L. F., Acevedo, L. G. O., et al. (2007). Optimisation of culture conditions for a synthetic gene expression in Escherichia coli using response surface methodology: The case of human interferon beta. Biomolecular Engineering, 24(2), 217–222. Scholar
  36. 36.
    Desvaux, M., Dumas, E., Chafsey, I., & Heébraud, M. (2006). Protein cell surface display in Gram-positive bacteria: From single protein to macromolecular protein structure. FEMS Microbiology Letters, 256, 1–15. Scholar
  37. 37.
    Vidová, B., Chotar, M., & Godány, A. (2009). N-terminal anchor in surface immunogenic protein of Streptococcus agalactiae and its influence on immunity elicitation. Folia Microbiologica, 54(2), 161–166. Scholar
  38. 38.
    Maione, D., Margarit, I., Rinaudo, C. D., Masignani, V., Mora, M., Scarselli, M., et al. (2005). Identification of a universal Group B streptococcus vaccine by multiple genome screen. Science, 309(5731), 148–150. Scholar
  39. 39.
    Baker, J. A., Lewis, E. L., Byland, L. M., Bonakdar, M., Randis, T. M., & Ratner, A. J. (2017). Mucosal vaccination promotes clearance of Streptococcus agalactiae vaginal colonisation. Vaccine, 35(9), 1273–1280. Scholar
  40. 40.
    Moyle, P. M. (2017). Biotechnology approaches to produce potent, self-adjuvanting antigen-adjuvant fusion protein subunit vaccines. Biotechnology Advances.Google Scholar
  41. 41.
    Lin, Z., Zhao, Q., Xing, L., Zhou, B., & Wang, X. (2015). Aggregating tags for column-free protein purification. Biotechnology Journal, 10(12), 1877–1886.CrossRefGoogle Scholar
  42. 42.
    Ratanji, K. D., Derrick, J. P., Kimber, I., Thorpe, R., Wadhwa, M., & Dearman, R. J. (2017). Influence of Escherichia coli chaperone DnaK on protein immunogenicity. Immunology, 150(3), 343–355.CrossRefGoogle Scholar
  43. 43.
    US Food and Drug Administration Guidance for industry. (2013). Immunogenicity assessment for therapeutic protein products. Rockville, MD: U.S. Department of Health and Human Services, FDA.Google Scholar

Copyright information

© Springer Science+Business Media, LLC, part of Springer Nature 2018

Authors and Affiliations

  • Diego A. Díaz-Dinamarca
    • 1
    • 2
  • José I. Jerias
    • 1
  • Daniel A. Soto
    • 1
  • Jorge A. Soto
    • 1
    • 2
  • Natalia V. Díaz
    • 1
  • Yessica Y. Leyton
    • 1
  • Rodrigo A. Villegas
    • 3
  • Alexis M. Kalergis
    • 2
    • 4
  • Abel E. Vásquez
    • 1
    • 5
    Email author
  1. 1.Sección BiotecnologíaInstituto de Salud Pública de ChileSantiagoChile
  2. 2.Departamento de Genética Molecular y Microbiología, Facultad de Ciencias BiológicasMillenium Institute on Immunology and ImmunotherapySantiagoChile
  3. 3.Departamento de Asuntos CientíficosInstituto de Salud Pública de ChileSantiagoChile
  4. 4.Departamento de Endocrinología, Facultad de Medicina PontificiaUniversidad Católica de ChileSantiagoChile
  5. 5.Facultad de CienciaUniversidad San SebastiánProvidenciaChile

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