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Protein Science (2005), 14:1729-1740. Published by Cold Spring Harbor Laboratory Press. Copyright © 2005 The Protein Society
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Design of improved membrane protein production experiments: Quantitation of the host response

Nicklas Bonander1,6, Kristina Hedfalk2,6, Christer Larsson2, Petter Mostad3, Celia Chang4, Lena Gustafsson2 and Roslyn M. Bill5

1 Department of Cell and Molecular Biology/Microbiology, Göteborg University, 405 30 Göteborg, Sweden
2 Department of Chemistry and Bioscience/Molecular Biotechnology, and 3 Department of Mathematics, Chalmers University of Technology, Göteborg, Sweden
4 The Wistar Institute, Philadelphia, Pennsylvania 19104, USA
5 School of Life and Health Sciences, Aston University, Birmingham B4 7ET, United Kingdom

(RECEIVED March 1, 2005; FINAL REVISION April 22, 2005; ACCEPTED April 22, 2005)

Eukaryotic membrane proteins cannot be produced in a reliable manner for structural analysis. Consequently, researchers still rely on trial-and-error approaches, which most often yield insufficient amounts. This means that membrane protein production is recognized by biologists as the primary bottleneck in contemporary structural genomics programs. Here, we describe a study to examine the reasons for successes and failures in recombinant membrane protein production in yeast, at the level of the host cell, by systematically quantifying cultures in high-performance bioreactors under tightly-defined growth regimes. Our data show that the most rapid growth conditions of those chosen are not the optimal production conditions. Furthermore, the growth phase at which the cells are harvested is critical: We show that it is crucial to grow cells under tightly-controlled conditions and to harvest them prior to glucose exhaustion, just before the diauxic shift. The differences in membrane protein yields that we observe under different culture conditions are not reflected in corresponding changes in mRNA levels of FPS1, but rather can be related to the differential expression of genes involved in membrane protein secretion and yeast cellular physiology.

Keywords: membrane proteins; expression systems; miniarray; protein production; yeast

Article and publication are at http://www.proteinscience.org/cgi/doi/10.1110/ps.051435705.

Reprint requests to: Roslyn M. Bill, School of Life and Health Sciences, Aston University, Aston Triangle, Birmingham B4 7ET, UK; e-mail: r.m.bill{at}aston.ac.uk; fax: +44-121-333-4220.


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