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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
Research

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.

Keywords

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

Notes

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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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

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