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Production of Membrane Proteins in Escherichia coli and Lactococcus lactis

  • Eric R. Geertsma
  • Bert PoolmanEmail author
Protocol
Part of the Methods in Molecular Biology™ book series (MIMB, volume 601)

Abstract

As the equivalent to gatekeepers of the cell, membrane transport proteins perform a variety of critical functions. Progress on the functional and structural characterization of membrane proteins is slowed due to problems associated with their (heterologous) overexpression. Often, overexpression fails or leads to aggregated material from which the production of functionally refolded protein is challenging. It is still difficult to predict whether a given membrane protein can be overproduced in a functional competent state. As a result, the most straightforward strategy to set up an overexpression system is to screen a multitude of conditions, including the comparison of homologues, type and location of (affinity) tags, and distinct expression hosts. Here, we detail methodology to rapidly establish and optimize (membrane) protein expression in Escherichia coli and Lactococcus lactis.

Key words

Arabinose promoter Escherichia coli folding indicator GFP Lactococcus lactis membrane proteins nisin-controlled expression overexpression PBAD 

Notes

Acknowledgments

We thank G.K. Schuurman-Wolters, M.B. Tol, M. Groeneveld, D.J. Slotboom, and S. Henstra for their contribution in establishing the methodologies discussed. This work was funded by the European Membrane Protein Consortium (grant 504601), a FEBS fellowship (to E.R.G.), and the Netherlands Proteomics Centre.

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Copyright information

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

Authors and Affiliations

  1. 1.Department of BiochemistryGroningen Biomolecular Sciences and Biotechnology Institute (GBB), Netherlands Proteomics Centre (NPC), and Zernike Institute for Advanced Materials, University of GroningenGroningenThe Netherlands

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