Chapter

Chapter 3.2 Expression and purification of membrane proteins for structural studies

Crystallography of biological macromolecules

Second Online Edition (2012)

Part 3. Techniques of molecular biology

  1. J. A. Ernst1,2,
  2. D. G. Yansura3,
  3. C. M. Koth4

Published Online: 14 APR 2012

DOI: 10.1107/97809553602060000811

International Tables for Crystallography

International Tables for Crystallography

How to Cite

Ernst, J. A., Yansura, D. G. and Koth, C. M. 2012. Expression and purification of membrane proteins for structural studies. International Tables for Crystallography. 92–98.

Author Information

  1. 1

    Department of Protein Chemistry, Genentech, 1 DNA Way, South San Francisco, California 94080, USA

  2. 2

    Department of Protein Engineering, Genentech, 1 DNA Way, South San Francisco, California 94080, USA

  3. 3

    Department of Antibody Engineering, Genentech, 1 DNA Way, South San Francisco, California 94080, USA

  4. 4

    Department of Structural Biology, Genentech, 1 DNA Way, South San Francisco, California 94080, USA

Publication History

  1. Published Online: 14 APR 2012

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Abstract

Despite the fact that over 200 unique membrane‐protein structures have now been solved, significant challenges remain at all stages of protein production for both structural and functional studies. First, recombinant expression levels of membrane proteins are typically very low. Second, target proteins must be solubilized from their native environment, the cell membrane, and purified. This requires the use of detergents that can also cause the protein to denature or aggregate. Indeed, the repertoire of detergents that have been used successfully for membrane‐protein structural studies is surprisingly limited. Third, obtaining well ordered crystals is often very difficult, even if milligramme quantities of high‐quality membrane protein can be obtained. Despite these and other barriers, significant progress has been made over the last several years in determining the structures of members of many challenging membrane‐protein families, including transporters, channels, intramembrane proteases and G‐protein‐coupled receptors. Based largely on these past successes, and analyses of recurrent trends, reasonable ‘first‐pass’ strategies can now be proposed for most membrane‐protein targets. Here, the current state of producing membrane proteins for structural studies is reviewed. An evidence‐guided approach is detailed that should provide reasonable starting points for the production of many membrane‐protein targets.

Keywords:

  • membrane proteins;
  • expression;
  • purification;
  • affinity tags