Molecular Biotechnology

, Volume 59, Issue 4–5, pp 117–127 | Cite as

Molecular Cloning, Structural Modeling and the Production of Soluble Triple-Mutated Diphtheria Toxoid (K51E/G52E/E148K) Co-expressed with Molecular Chaperones in Recombinant Escherichia coli

  • Naphatsamon Uthailak
  • Pornpimol Mahamad
  • Pamorn Chittavanich
  • Somchai Yanarojana
  • Wassana Wijagkanalan
  • Jean Petre
  • Watanalai Panbangred
Original Paper


CRM197 is a diphtheria toxin (DT) mutant (G52E) which has been used as a carrier protein for conjugate vaccines. However, it still possesses cytotoxicity toward mammalian cells. The goal of this project was to produce a non-toxic and soluble CRM197EK through introduction of triple amino acid substitutions (K51E/G52E/E148K) in Escherichia coli. The expression of CRM197EKTrxHis was optimized and co-expressed with different molecular chaperones. The soluble CRM197EKTrxHis was produced at a high concentration (97.33 ± 17.47 μg/ml) under the optimal condition (induction with 0.1 mM IPTG at 20 °C for 24 h). Cells containing pG-Tf2, expressing trigger factor and GroEL-GroES, accumulated the highest amount of soluble CRM197EKTrxHis at 111.24 ± 10.40 μg/ml after induction for 24 h at 20 °C. The soluble CRM197EKTrxHis still possesses nuclease activity and completely digest λDNA at 25 and 37 °C with 8- and 4-h incubation, respectively. Molecular modeling of diphtheria toxin, CRM197 and CRM197EK indicated that substitutions of two amino acids (K51E/E148K) may cause poor NAD binding, consistent with the lack of toxicity. Therefore, CRM197EK might be used as a new potential carrier protein. However, further in vivo study is required to confirm its roles as functional carrier protein in conjugate vaccines.


Cross-reacting material 197 CRM197EK Escherichia coli Molecular chaperones Molecular modeling 


  1. 1.
    Uchida, T., Pappenheimer, A., & Greany, R. (1973). Diphtheria toxin and related proteins I. Isolation and properties of mutant proteins serologically related to diphtheria toxin. Journal of Biological Chemistry, 248, 3838–3844.Google Scholar
  2. 2.
    Deng, Q., & Barbieri, J. (2008). Molecular mechanisms of the cytotoxicity of ADP-ribosylating toxins. Annual Review of Microbiology, 62, 271–288.CrossRefGoogle Scholar
  3. 3.
    Bell, C., & Eisenberg, D. (1996). Crystal structure of diphtheria toxin bound to nicotinamide adenine dinucleotide. Biochemistry, 35, 1137–1149.CrossRefGoogle Scholar
  4. 4.
    Pappenheimer, J. A. (1977). Diphtheria toxin. Annual Review of Biochemistry, 46, 69–94.CrossRefGoogle Scholar
  5. 5.
    Shinefield, H. (2010). Overview of the development and current use of CRM 197 conjugate vaccines for pediatric use. Vaccine, 28, 4335–4339.CrossRefGoogle Scholar
  6. 6.
    Bishai, W., Rappuoli, R., & Murphy, J. (1987). High-level expression of a proteolytically sensitive diphtheria toxin fragment in Escherichia coli. Journal of Bacteriology, 169, 5140–5151.CrossRefGoogle Scholar
  7. 7.
    Jianhua, Z., & Petracca, R. (1999). Secretory expression of recombinant diphtheria toxin mutants in Bacillus subtilis. Journal of Tongji Medical University, 19, 253–256.CrossRefGoogle Scholar
  8. 8.
    Mahamad, P., Boonchird, C., & Panbangred, W. (2016). High level accumulation of soluble diphtheria toxin mutant (CRM197) with co-expression of chaperones in recombinant Escherichia coli. Applied Microbiology and Biotechnology, 100, 6319–6330.CrossRefGoogle Scholar
  9. 9.
    Malito, E., Bursulaya, B., Chen, C., Surdo, P., Picchianti, M., Balducci, E., et al. (2012). Structural basis for lack of toxicity of the diphtheria toxin mutant CRM197. Proceedings of the National Academy of Sciences of the United States of America, 109, 5229–5234.CrossRefGoogle Scholar
  10. 10.
    Kimura, Y., Saito, M., Kimata, Y., & Kohno, K. (2007). Transgenic mice expressing a fully nontoxic diphtheria toxin mutant, not CRM197 mutant, acquire immune tolerance against diphtheria toxin. Journal of Biochemistry, 142, 105–112.CrossRefGoogle Scholar
  11. 11.
    Kageyama, T., Ohishi, M., Miyamoto, S., Mizushima, H., Iwamoto, R., & Mekada, E. (2007). Diphtheria toxin mutant CRM197 possesses weak EF2-ADP-ribosyl activity that potentiates its anti-tumorigenic activity. Journal of Biochemistry, 142, 95–104.CrossRefGoogle Scholar
  12. 12.
    Qiao, J., Ghani, K., & Caruso, M. (2008). Diphtheria toxin mutant CRM197 is an inhibitor of protein synthesis that induces cellular toxicity. Toxicon, 51, 473–477.CrossRefGoogle Scholar
  13. 13.
    Bell, C., & Eisenberg, D. (1997). Crystal structure of nucleotide-free diphtheria toxin. Biochemistry, 36, 481–488.CrossRefGoogle Scholar
  14. 14.
    Schwede, T., Kopp, J., Guex, N., & Peitsch, M. (2003). SWISS-MODEL: An automated protein homology-modeling server. Nucleic Acids Research, 31, 3381–3385.CrossRefGoogle Scholar
  15. 15.
    Case, D., Darden, T., Cheatham, T., Simmerling, C., Wang, J., Duke, R., et al. (2012). AMBER 12. San Francisco: University of California.Google Scholar
  16. 16.
    Duan, Y., Wu, C., Chowdhury, S., Lee, M., Xiong, G., Zhang, W., et al. (2003). A point-charge force field for molecular mechanics simulations of proteins based on condensed-phase quantum mechanical calculations. Journal of Computational Chemistry, 24, 1999–2012.CrossRefGoogle Scholar
  17. 17.
    Berendsen, H., Postma, J., Gunsteren, W., DiNola, A., & Haak, J. (1984). Molecular dynamics with coupling to an external bath. The Journal of Chemical Physics, 81, 3684–3690.CrossRefGoogle Scholar
  18. 18.
    Essmann, U., Perera, L., Berkowitz, M., Darden, T., Lee, H., & Pedersen, L. (1995). A smooth particle mesh Ewald method. The Journal of Chemical Physics, 103, 8577–8593.CrossRefGoogle Scholar
  19. 19.
    Prinz, W., Åslund, F., Holmgren, A., & Beckwith, J. (1997). The role of the thioredoxin and glutaredoxin pathways in reducing protein disulfide bonds in the Escherichia coli cytoplasm. Journal of Biological Chemistry, 272, 15661–15667.CrossRefGoogle Scholar
  20. 20.
    Rabhi-Essafi, I., Sadok, A., Khalaf, N., & Fathallah, D. (2007). A strategy for high-level expression of soluble and functional human interferon α as a GST-fusion protein in Escherichia coli. Protein Engineering, Design & Selection, 20, 201–209.CrossRefGoogle Scholar
  21. 21.
    Studier, F., Daegelen, P., Lenski, R., Maslov, S., & Kim, J. (2009). Understanding the differences between genome sequences of Escherichia coli B strains REL606 and BL21 (DE3) and comparison of the Escherichia coli B and K-12 genomes. Journal of Molecular Biology, 394, 653–680.CrossRefGoogle Scholar
  22. 22.
    Joseph, S., & David, W. (2001). Molecular cloning: a laboratory manual. New York: Gold Spring Harbor.Google Scholar
  23. 23.
    Vera, A., González-Montalbán, N., Arís, A., & Villaverde, A. (2007). The conformational quality of insoluble recombinant proteins is enhanced at low growth temperatures. Biotechnology and Bioengineering, 96, 1101–1106.CrossRefGoogle Scholar
  24. 24.
    Hendrick, J., & Hartl, F. (1993). Molecular chaperone functions of heat-shock proteins. Annual Review of Biochemistry, 62, 349–384.CrossRefGoogle Scholar
  25. 25.
    Weickert, M., Doherty, D., Best, E., & Olins, P. (1996). Optimization of heterologous protein production in Escherichia coli. Current Opinion in Biotechnology, 7, 494–499.CrossRefGoogle Scholar
  26. 26.
    Nishihara, K., Kanemori, M., Yanagi, H., & Yura, T. (2000). Overexpression of trigger factor prevents aggregation of recombinant proteins in Escherichia coli. Applied and Environmental Microbiology, 66, 884–889.CrossRefGoogle Scholar
  27. 27.
    Rosano, G. L., & Ceccarelli, E. A. (2014). Recombinant protein expression in Escherichia coli: Advances and challenges. Frontiers in Microbiology, 5, 172.Google Scholar
  28. 28.
    Kohanski, M., Dwyer, D., & Collins, J. (2010). How antibiotics kill bacteria: from targets to networks. Nature Reviews Microbiology, 8, 423–435.CrossRefGoogle Scholar
  29. 29.
    Martínez-Alonso, M., García-Fruitós, E., Ferrer-Miralles, N., Rinas, U., & Villaverde, A. (2010). Side effects of chaperone gene co-expression in recombinant protein production. Microbial Cell Factories, 9, 1.CrossRefGoogle Scholar
  30. 30.
    Jia, Q., Fan, D., Ma, P., Ma, X., & Xue, W. (2014). The different roles of chaperone teams on over-expression of human-like collagen in recombinant Escherichia coli. Journal of the Taiwan Institute of Chemical Engineers, 45, 2843–2850.CrossRefGoogle Scholar
  31. 31.
    Bruce, C., Baldwin, R., Lessnick, S., & Wisnieski, B. (1990). Diphtheria toxin and its ADP-ribosyltransferase-defective homologue CRM197 possess deoxyribonuclease activity. Proceedings of the National Academy of Sciences, 87, 2995–2998.CrossRefGoogle Scholar
  32. 32.
    Stefan, A., Conti, M., Rubboli, D., Ravagli, L., Presta, E., & Hochkoeppler, A. (2011). Overexpression and purification of the recombinant diphtheria toxin variant CRM197 in Escherichia coli. Journal of Biotechnology, 156, 245–252.CrossRefGoogle Scholar
  33. 33.
    Perera, V., & Corbel, M. (1990). Human antibody response to fragments A and B of diphtheria toxin and a synthetic peptide of amino acid residues 141–157 of fragment A. Epidemiology and Infection, 105, 457–468.CrossRefGoogle Scholar
  34. 34.
    Raju, R., Navaneetham, D., Okita, D., Diethelm-Okita, B., McCormick, D., & Conti-Fine, B. (1995). Epitopes for human CD4 + cells on diphtheria toxin: Structural features of sequence segments forming epitopes recognized by most subjects. European Journal of Immunology, 25, 3207–3214.CrossRefGoogle Scholar
  35. 35.
    Bröker, M., Costantino, P., DeTora, L., McIntosh, E., & Rappuoli, R. (2011). Biochemical and biological characteristics of cross-reacting material 197 (CRM197), a non-toxic mutant of diphtheria toxin: Use as a conjugation protein in vaccines and other potential clinical applications. Biologicals, 39, 195–204.CrossRefGoogle Scholar
  36. 36.
    Pecetta, S., Vijayakrishnan, B., Romano, M., Proietti, D., Surdo, P., Balocchi, C., et al. (2016). Evaluation of the non-toxic mutant of the diphtheria toxin K51E/E148 K as carrier protein for meningococcal vaccines. Vaccine, 34, 1405–1411.CrossRefGoogle Scholar
  37. 37.
    Mignon, C., Sodoyer, R., & Werle, B. (2015). Antibiotic-free selection in biotherapeutics: Now and forever. Pathogens, 4(2), 157–181.CrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media New York 2017

Authors and Affiliations

  1. 1.Department of Biotechnology, Faculty of ScienceMahidol UniversityBangkokThailand
  2. 2.Mahidol University - Osaka University Collaborative Research Center for Bioscience and Biotechnology (MU-OU: CRC), Faculty of ScienceMahidol UniversityBangkokThailand
  3. 3.Department of Pharmacology, Faculty of ScienceMahidol UniversityBangkokThailand
  4. 4.BioNet-Asia Co., Ltd., 19 Udomsuk 37BangkokThailand

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