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Membrane Protein Expression in Lactococcus lactis

  • Annie Frelet-BarrandEmail author
  • Sylvain Boutigny
  • Edmund R. S. Kunji
  • Norbert Rolland
Protocol
Part of the Methods in Molecular Biology™ book series (MIMB, volume 601)

Abstract

Membrane proteins play key roles in cellular physiology, and they are important drug targets. Approximately 25% of all genes identified in sequenced genomes are known to encode membrane proteins; however, the majority have no assigned function. Although the resolution of soluble protein structure has entered the high-throughput stage, only 100 high-resolution structures of membrane proteins have been described until now. Lactococcus lactis is a gram-positive lactic bacterium that has been used traditionally in food fermentations, but it is now used widely in biotechnology for large-scale overproduction of heterologously expressed proteins. Various expression vectors based on either constitutive or inducible promoters exist. The nisin-inducible controlled gene expression (NICE) system is the most suitable for recombinant membrane protein expression allowing for fine control of gene expression based on the autoregulation mechanism of the bacteriocin nisin. Recombinant membrane proteins can be produced with affinity tags for efficient detection and purification from crude membrane protein extracts. The purpose of this chapter is to provide a detailed protocol for the expression of membrane proteins and their detection using the Strep-tag II affinity tag in L. lactis.

Key words

Cell disruptor French press Lactococcus lactis membrane proteins Strep-tag II 

Notes

Acknowledgments

This work has been supported by the Commissariat à l’Energie Atomique (CEA-PM project, A.F., S.B., and N.R.). We thank Shaun Peters and Daphné Seigneurin-Berny for critical reading of the manuscript. We thank Igor Mierau (NIZO, The Netherlands) for kindly allowing us to illustrate this chapter with the figure on the NICE system.

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Copyright information

© Humana Press, a part of Springer Science+Business Media, LLC 2010

Authors and Affiliations

  • Annie Frelet-Barrand
    • 1
    Email author
  • Sylvain Boutigny
    • 2
    • 3
    • 4
    • 5
  • Edmund R. S. Kunji
    • 6
  • Norbert Rolland
    • 3
    • 4
    • 5
    • 7
  1. 1.Laboratoire de Physiologie Cellulaire Végétale, CNRS (UMR-5168)/CEA/INRA (UMR-1200), Université Joseph Fourier, iRTSVCEA-GrenobleFrance
  2. 2.Laboratoire de Physiologie Cellulaire VégétaleGrenobleFrance
  3. 3.CEA, DSV, iRTSVGrenobleFrance
  4. 4.INRAGrenobleFrance
  5. 5.Université Joseph FourierGrenobleFrance
  6. 6.The Medical Research Council, Dunn Human Nutrition UnitCambridgeUK
  7. 7.Laboratoire de Physiologie Cellulaire Végétale, CNRSGrenobleFrance

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