Molecular Biotechnology

, Volume 55, Issue 3, pp 227–235 | Cite as

An Improved Strategy for Easy Process Monitoring and Advanced Purification of Recombinant Proteins

  • Baligh Miladi
  • Cyrine Dridi
  • Ahmed El Marjou
  • Guilhem Boeuf
  • Hassib Bouallagui
  • Florence Dufour
  • Patrick Di Martino
  • Abdellatif Elm’selmi


In this work, a multifunctional expression cassette, termed Multitags, combining different and complementary functionalities, was designed and used to monitor the expression and the purification of two model proteins (Pfu DNA polymerase and Myosin-VIIa- and Rab-Interracting protein : MyRIP). Multitags contains two affinity purification tags, a polyhistidine sequence (10× His) and the streptavidin-binding peptide (SBP) and as a marker tag the heme-binding domain of rat cytochrome b5 followed by the TEV cleavage site. Using the Multitags as fusion partner, more than 90 % of both fusion proteins were produced in soluble form when expressed in Escherichia coli KRX. In addition, high purity (99 %) of recombinant proteins was achieved after two consecutive affinity purification steps. The expression cassette also demonstrated an accurate monitoring capability comparable to that of a dual recognition-based method. The choice of the SBP tag was considered as an integral process that included a method for tag removal. Thus, an immobilized TEV protease fixed on streptavidin–agarose matrix was used for the cleavage of fusion proteins. After digestion, both unprocessed fusion proteins and Multitags were retained on the proteolytic column via their SBP sequence, allowing cleavage and recovery of target proteins on one step. This combined approach may accelerate the development of optimized production processes, while insuring high product quality and a low production cost.


Process monitoring Cytochrome b5 SBP tag 10× His tag Double purification Recombinant protein 



This study was supported by the public company OSEO ( We wish to thank Ms. Nabila Hocine for her technical support.


  1. 1.
    Esposito, D., & Chatterjee, D. K. (2006). Enhancement of soluble protein expression through the use of fusion tags. Current Opinion in Biotechnology, 17, 353–358.CrossRefGoogle Scholar
  2. 2.
    Arnau, J., Lauritzen, C., Petersen, G. E., & Pedersen, J. (2006). Current strategies for the use of affinity tags and tag removal for the purification of recombinant proteins. Protein Expression and Purification, 48, 1–13.CrossRefGoogle Scholar
  3. 3.
    Waugh, D. S. (2005). Making the most of affinity tags. Trends in Biotechnology, 23, 316–320.CrossRefGoogle Scholar
  4. 4.
    Malhotra, A. (2009). Tagging for protein expression. Methods in Enzymology, 463, 239–258.CrossRefGoogle Scholar
  5. 5.
    Clementschitsch, F., & Bayer, K. (2006). Improvements of bioprocess monitoring: Development of novel concepts. Microbial Cell Factories, 5, 19.CrossRefGoogle Scholar
  6. 6.
    Vojinović, V., Cabral, J. M. S., & Fonseca, L. P. (2006). Real-time bioprocess monitoring: Part I: In situ sensors. Sensors and Actuators, B, 114, 1083–1091.CrossRefGoogle Scholar
  7. 7.
    Albano, C. R., Randers-Eichhorn, L., Bentley, W. E., & Rao, G. (1998). Green fluorescent protein as a real time quantitative reporter of heterotopous protein production. Biotechnology Progress, 14, 350–354.CrossRefGoogle Scholar
  8. 8.
    Reischer, H., Schotola, I., Striedner, G., Pötschacher, F., & Bayer, K. (2004). Evaluation of the GFP signal and its aptitude for novel on-line monitoring strategies of recombinant fermentation processes. Journal of Biotechnology, 108, 115–125.CrossRefGoogle Scholar
  9. 9.
    Chae, H. J., DeLisa, M. P., Cha, H. Y., Weingand, W. A., Rao, G., & Bentley, W. E. (2000). Framework for on-line optimisation of recombinant protein expression in high cell density Escherichia coli cultures using GFP-fusion monitoring. Biotechnology and Bioengineering, 69, 275–285.CrossRefGoogle Scholar
  10. 10.
    Jones, J. J., Bridges, A. M., Fosberry, A. P., Gardner, S., Lowers, R. R., Newby, R. R., et al. (2004). Potential of real-time measurement of GFP-fusion proteins. Journal of Biotechnology, 109, 201–211.CrossRefGoogle Scholar
  11. 11.
    Finn, R. D., Kapelioukh, I., & Paine, M. J. I. (2005). Rainbow tags: A visual tag system for recombinant protein expression and purification. BioTechniques, 38, 387–392.CrossRefGoogle Scholar
  12. 12.
    Surribas, A., Resina, D., Ferrer, P., & Valero, F. (2007). Rivoflavin may interfere with on-line monitoring of secreted green fluorescence protein fusion proteins in Pichia pastoris. Microbial Cell Factories, 6, 15.CrossRefGoogle Scholar
  13. 13.
    Kohli, B. M., & Ostermeier, C. (2003). A Rubredoxin based system for screening of protein expression conditions and on-line monitoring of the purification process. Protein Expression and Purification, 28, 362–367.CrossRefGoogle Scholar
  14. 14.
    Mitra, A., Chakrabarti, K. S., Shahul Hameed, M. S., Srinivas, K. V., Senthil Kumar, G., & Sarma, S. P. (2005). High level expression of peptides and proteins using cytochrome b5 as a fusion host. Protein Expression and Purification, 41, 84–97.CrossRefGoogle Scholar
  15. 15.
    Cass, B., Pham, P. L., Kamen, A., & Durocher, Y. (2005). Purification of recombinant proteins from mammalian cell culture using a generic double-affinity chromatography scheme. Protein Expression and Purification, 40, 77–85.CrossRefGoogle Scholar
  16. 16.
    Prinz, B., Schultchen, J., Rydzewski, R., Holz, C., Boettner, M., Stahl, U., et al. (2004). Establishing a versatile fermentation and purification procedure for human proteins expressed in the yeasts Saccharomyces cerevisiae and Pichia pastoris for structural genomics. Journal of Structural and Functional Genomics, 5, 29–44.CrossRefGoogle Scholar
  17. 17.
    Collins, M. O., & Choudhary, J. S. (2008). Mapping multiprotein complexes by affinity purification and mass spectrometry. Current Opinion in Biotechnology, 19, 324–330.CrossRefGoogle Scholar
  18. 18.
    Li, Y. (2010). Commonly used tag combinations for tandem affinity purification. Biotechnology and Applied Biochemistry, 55, 73–83.CrossRefGoogle Scholar
  19. 19.
    Li, Y., Franklin, S., Zhang, M. J., & Vondriska, T. M. (2011). Highly efficient purification of protein complexes from mammalian cells using a novel streptavidin-binding peptide and hexahistidine tandem tag system: Application to Bruton’s tyrosine kinase. Protein Science, 20, 140–149.CrossRefGoogle Scholar
  20. 20.
    Nguyen, H., Martinez, B., Oganesyan, N., & Kim, R. (2004). An automated small-scale protein expression and purification screening provides beneficial information for protein production. Journal of Structural and Functional Genomics, 5, 23–27.CrossRefGoogle Scholar
  21. 21.
    Sun, P., Tropea, J. E., & Waugh, D. S. (2011). Enhancing the solubility of recombinant proteins in Escherichia coli by using hexahistidine-tagged maltose-binding protein as a fusion partner. Methods in Molecular Biology, 705, 259–274.CrossRefGoogle Scholar
  22. 22.
    Liu, H., & Naismith, J. H. (2009). A simple and efficient expression and purification system using two newly constructed vectors. Protein Expression and Purification, 63, 102–111.CrossRefGoogle Scholar
  23. 23.
    Miladi, B. El., Marjou, A., Bœuf, G., Bouallagui, H., Di Dufour, F., Martino, P., et al. (2012). Oriented immobilization of the tobacco etch virus protease for the cleavage of fusion proteins. Journal of Biotechnology, 158, 97–103.CrossRefGoogle Scholar
  24. 24.
    Miladi, B., Bouallagui, H., El. Dridi, C., Marjou, A., Di. Boeuf, G., Martino, P., et al. (2011). A new tagged-TEV protease: Construction, optimisation of production, purification and test activity. Protein Expression and Purification, 75, 75–82.CrossRefGoogle Scholar
  25. 25.
    Davis, G. D., Elisee, C., Newham, D. M., & Harrison, R. G. (1999). New fusion protein systems designed to give soluble expression in Escherichia coli. Biotechnology and Bioengineering, 65, 382–388.CrossRefGoogle Scholar
  26. 26.
    Lin, Y. W., Zhao, D. X., Wang, Z. H., Yu, W. H., & Huang, Z. X. (2006). Expression of lipase-solubilized bovine liver microsomal cytochrome b5 in Escherichia coli as a glutathione S-transferase fusion protein (GST-cyt b 5). Protein Expression and Purification, 45, 352–358.CrossRefGoogle Scholar
  27. 27.
    Ellis, J., Gutierrez, A., Barsukov, I. L., Huang, W. C., Grossmann, J. G., & Roberts, G. C. (2009). Domain motion in cytochrome P450 reductase: Conformational equilibria revealed by NMR and small-angle X-ray scattering. Journal of Biological Chemistry, 284, 36628–36637.CrossRefGoogle Scholar
  28. 28.
    Kobayashi, T., Morone, N., Kashiyama, T., Oyamada, H., Kurebayashi, N., & Murayama, T. (2008). Engineering a novel multifunctional green fluorescent protein tag for a wide variety of protein research. PLoS One, 3, e3822.CrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media New York 2013

Authors and Affiliations

  • Baligh Miladi
    • 1
    • 4
  • Cyrine Dridi
    • 1
  • Ahmed El Marjou
    • 2
  • Guilhem Boeuf
    • 1
  • Hassib Bouallagui
    • 3
  • Florence Dufour
    • 1
  • Patrick Di Martino
    • 4
  • Abdellatif Elm’selmi
    • 1
  1. 1.Laboratoire de Biologie MoléculaireEcole de Biologie IndustrielleCergy-Pontoise cedexFrance
  2. 2.Plateforme de production d’anticorps et de protéines recombinantesInstitut Curie/CNRS UMR144Paris Cedex 5France
  3. 3.Laboratoire d’Ecologie et de Technologie Microbienne, INSATTunis CedexTunisia
  4. 4.Laboratoire ERRMECe (EA1391)Université de Cergy-PontoiseCergy-Pontoise CedexFrance

Personalised recommendations