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J Mol Microbiol Biotechnol 2010;18:85–91
(DOI:10.1159/000283512)

An Escherichia coli-Based Cell-Free System for Large-Scale Production of Functional Mammalian Membrane Proteins Suitable for X-Ray Crystallography

Nguyen T.A. · Lieu S.S. · Chang G.
Department of Molecular Biology, The Scripps Research Institute, La Jolla, Calif., USA
email Corresponding Author


 goto top of outline Key Words

  • Cell-free expression
  • Escherichia coli
  • Membrane protein
  • X-ray structural analysis

 goto top of outline Abstract

A cell-free expression system using an Escherichia coli extract was adapted for large-scale expression and purification of mammalian membrane proteins. The system was tested with a set of human membrane proteins of different sizes, numbers of transmembrane domains, oligomeric arrangements, and native membrane locations. Tens of milligrams of protein were readily expressed and purified from an overnight cell-free reaction. Both reaction ‘mode A’ (proteins were expressed as precipitant) and ‘mode B’ (proteins were expressed in the presence of mild detergents to keep them soluble) were investigated. The combination of ‘mode B’ and the right detergents, used in the subsequent extraction and purification steps, is critical for obtaining properly folded proteins (CX32 and VDAC1) that can be crystallized and diffracted (VDAC1). The E. coli cell-free system is capable of efficient expression of many mammalian membrane proteins. However, fine-tuning of the system, especially to facilitate proper protein folding, will be required for each specific target.

Copyright © 2010 S. Karger AG, Basel


 goto top of outline References
  1. Bayrhuber M, Meins T, Habeck M, Becker S, Giller K, Villinger S, Vonrhein C, Griesinger C, Zweckstetter M, Zeth K: Structure of the human voltage-dependent anion channel. Proc Natl Acad Sci USA 2008;105:15370–15375.
  2. Berrier C, Park KH, Abes S, Bibonne A, Betton JM, Ghazi A: Cell-free synthesis of a functional ion channel in the absence of a membrane and in the presence of detergent. Biochemistry 2004;43:12585–12591.
  3. Chen YJ, Pornillos O, Lieu S, Ma C, Chen AP, Chang G: X-ray structure of EmrE supports dual topology model. Proc Natl Acad Sci USA 2007;104:18999–19004.
  4. Elbaz Y, Steiner-Mordoch S, Danieli T, Schuldiner S: In vitro synthesis of fully functional EmrE, a multidrug transporter, and study of its oligomeric state. Proc Natl Acad Sci USA 2004;101:1519–1524.
  5. He M: Cell-free protein synthesis: applications in proteomics and biotechnology. N Biotechnol 2008;25:126–132.
  6. Hiller S, Garces RG, Malia TJ, Orekhov VY, Colombini M, Wagner G: Solution structure of the integral human membrane protein VDAC-1 in detergent micelles. Science 2008;321:1206–1210.
  7. Katzen F, Chang G, Kudlicki W: The past, present and future of cell-free protein synthesis. Trends Biotechnol 2005;23:150–156.
  8. Kigawa T, Yabuki T, Yoshida Y, Tsutsui M, Ito Y, Shibata T, Yokoyama S: Cell-free production and stable-isotope labeling of milligram quantities of proteins. FEBS Lett 1999;442:15–19.
  9. Klammt C, Schwarz D, Eifler N, Engel A, Piehler J, Haase W, Hahn S, Dotsch V, Bernhard F: Cell-free production of G protein-coupled receptors for functional and structural studies. J Struct Biol 2007;158:482–493.
  10. Klammt C, Schwarz D, Lohr F, Schneider B, Dotsch V, Bernhard F: Cell-free expression as an emerging technique for the large scale production of integral membrane protein. FEBS J 2006;273:4141–4153.
  11. Maeda S, Nakagawa S, Suga M, Yamashita E, Oshima A, Fujiyoshi Y, Tsukihara T: Structure of the connexin 26 gap junction channel at 3.5 Å resolution. Nature 2009;458:597–602.
  12. Morita EH, Sawasaki T, Tanaka R, Endo Y, Kohno T: A wheat germ cell-free system is a novel way to screen protein folding and function. Protein Sci 2003;12:1216–1221.
  13. Nirenberg MW, Matthaei JH: The dependence of cell-free protein synthesis in E. coli upon naturally occurring or synthetic polyribonucleotides. Proc Natl Acad Sci USA 1961;47:1588–1602.
  14. Nozawa A, Nanamiya H, Miyata T, Linka N, Endo Y, Weber AP, Tozawa Y: A cell-free translation and proteoliposome reconstitution system for functional analysis of plant solute transporters. Plant Cell Physiol 2007;48:1815–1820.
  15. Shao L, Kinnally KW, Mannella CA: Circular dichroism studies of the mitochondrial channel, VDAC, from Neurospora crassa. Biophys J 1996;71:778–786.
  16. Shimizu Y, Inoue A, Tomari Y, Suzuki T, Yokogawa T, Nishikawa K, Ueda T: Cell-free translation reconstituted with purified components. Nat Biotechnol 2001;19:751–755.
  17. Ujwal R, Cascio D, Colletier JP, Faham S, Zhang J, Toro L, Ping P, Abramson J: The crystal structure of mouse VDAC1 at 2.3 A resolution reveals mechanistic insights into metabolite gating. Proc Natl Acad Sci USA 2008;105:17742–17747.
  18. Zhang Q, Horst R, Geralt M, Ma X, Hong WX, Finn MG, Stevens RC, Wuthrich K: Microscale NMR screening of new detergents for membrane protein structural biology. J Am Chem Soc 2008;130:7357–7363.
  19. Zhang Q, Ma X, Ward A, Hong WX, Jaakola VP, Stevens RC, Finn MG, Chang G: Designing facial amphiphiles for the stabilization of integral membrane proteins. Angew Chem Int Ed Engl 2007;46:7023–7025.

 goto top of outline Author Contacts

Geoffrey Chang
Department of Molecular Biology, The Scripps Research Institute
10550 North Torrey Pines Road
La Jolla, CA 92037 (USA)
Tel. +1 858 784 9490, Fax +1 858 784 9985, E-Mail gchang@scripps.edu


 goto top of outline Article Information

Received and accepted: October 29, 2009
Published online: February 17, 2010
Number of Print Pages : 7
Number of Figures : 5, Number of Tables : 1, Number of References : 19


 goto top of outline Publication Details

Journal of Molecular Microbiology and Biotechnology

Vol. 18, No. 2, Year 2010 (Cover Date: April 2010)

Journal Editor: Saier Jr. M.H. (La Jolla, Calif.)
ISSN: 1464-1801 (Print), eISSN: 1660-2412 (Online)

For additional information: http://www.karger.com/MMB


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