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

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Abstract

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.

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References

  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.

    Article  CAS  Google Scholar 

  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.

    Article  CAS  Google Scholar 

  3. Waugh, D. S. (2005). Making the most of affinity tags. Trends in Biotechnology, 23, 316–320.

    Article  CAS  Google Scholar 

  4. Malhotra, A. (2009). Tagging for protein expression. Methods in Enzymology, 463, 239–258.

    Article  CAS  Google Scholar 

  5. Clementschitsch, F., & Bayer, K. (2006). Improvements of bioprocess monitoring: Development of novel concepts. Microbial Cell Factories, 5, 19.

    Article  Google Scholar 

  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.

    Article  Google Scholar 

  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.

    Article  Google Scholar 

  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.

    Article  CAS  Google Scholar 

  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.

    Article  CAS  Google Scholar 

  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.

    Article  CAS  Google Scholar 

  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.

    Article  CAS  Google Scholar 

  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.

    Article  Google Scholar 

  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.

    Article  CAS  Google Scholar 

  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.

    Article  CAS  Google Scholar 

  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.

    Article  CAS  Google Scholar 

  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.

    Article  CAS  Google Scholar 

  17. Collins, M. O., & Choudhary, J. S. (2008). Mapping multiprotein complexes by affinity purification and mass spectrometry. Current Opinion in Biotechnology, 19, 324–330.

    Article  CAS  Google Scholar 

  18. Li, Y. (2010). Commonly used tag combinations for tandem affinity purification. Biotechnology and Applied Biochemistry, 55, 73–83.

    Article  CAS  Google Scholar 

  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.

    Article  Google Scholar 

  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.

    Article  CAS  Google Scholar 

  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.

    Article  CAS  Google Scholar 

  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.

    Article  CAS  Google Scholar 

  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.

    Article  CAS  Google Scholar 

  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.

    Article  CAS  Google Scholar 

  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.

    Article  CAS  Google Scholar 

  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.

    Article  CAS  Google Scholar 

  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.

    Article  CAS  Google Scholar 

  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.

    Article  Google Scholar 

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Acknowledgments

This study was supported by the public company OSEO (http://www.oseo.fr). We wish to thank Ms. Nabila Hocine for her technical support.

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Authors and Affiliations

  1. Laboratoire de Biologie Moléculaire, Ecole de Biologie Industrielle, 32 Boulevard du port, 95094, Cergy-Pontoise cedex, France

    Baligh Miladi, Cyrine Dridi, Guilhem Boeuf, Florence Dufour & Abdellatif Elm’selmi

  2. Plateforme de production d’anticorps et de protéines recombinantes, Institut Curie/CNRS UMR144, 26 rue d’Ulm, 75248, Paris Cedex 5, France

    Ahmed El Marjou

  3. Laboratoire d’Ecologie et de Technologie Microbienne, INSAT, Centre Urbain Nord BP 676, 1080, Tunis Cedex, Tunisia

    Hassib Bouallagui

  4. Laboratoire ERRMECe (EA1391), Université de Cergy-Pontoise, 2 Avenue Adolphe-Chauvin, 95302, Cergy-Pontoise Cedex, France

    Baligh Miladi & Patrick Di Martino

Authors
  1. Baligh Miladi
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  2. Cyrine Dridi
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  3. Ahmed El Marjou
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  4. Guilhem Boeuf
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  5. Hassib Bouallagui
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  6. Florence Dufour
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  7. Patrick Di Martino
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  8. Abdellatif Elm’selmi
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Correspondence to Abdellatif Elm’selmi.

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Miladi, B., Dridi, C., El Marjou, A. et al. An Improved Strategy for Easy Process Monitoring and Advanced Purification of Recombinant Proteins. Mol Biotechnol 55, 227–235 (2013). https://doi.org/10.1007/s12033-013-9673-5

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  • DOI: https://doi.org/10.1007/s12033-013-9673-5

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