The long-term objectives of this proposal are to establish generic techniques for membrane protein production and purification in different hosts and in formats that scale to high-throughput environments. Specifically, the GFP-based expression/purification pipeline recently set up for E. coli will be further developed (including a new detergent solubilization screen and improved vectors), and will also be implemented for the yeasts Saccharomyces cerevisiae and Pichia pastoris. A selection of S. cerevisiae membrane proteins will be put through the GFP pipeline in both hosts, and promising targets will be delivered for crystallization trials. The response of E. coli cells to overexpression of a set of bacterial and eukaryotic membrane proteins will be characterized by proteomics techniques, and strain engineering will be carried out based on the results obtained. Finally, a large number of E. coli operons containing predicted membrane proteins will be cloned and both homo- and hetero-oligomeric complexes will be identified by blue-native/SDS-PAGE analysis. Well-expressing complexes will be put through the GFP-based pipeline, and promising candidates will be delivered to crystallization trials. Relevance: Proper functioning of the cell membrane in terms of the import and export of molecules, the sending and receiving of signals, and the interactions with neighboring cells are absolutely essential to the health of the organism. Most of these functions are carried out by proteins in the cell membrane, yet, such proteins are difficult to produce and study biochemically. The project aims to develop better techniques for producing membrane proteins on a large scale. ? ? ?
| Hjelm, Anna; Schlegel, Susan; Baumgarten, Thomas et al. (2013) Optimizing E. coli-based membrane protein production using Lemo21(DE3) and GFP-fusions. Methods Mol Biol 1033:381-400 |
| Norholm, Morten H H; Toddo, Stephen; Virkki, Minttu T I et al. (2013) Improved production of membrane proteins in Escherichia coli by selective codon substitutions. FEBS Lett 587:2352-8 |
| Schlegel, Susan; Rujas, Edurne; Ytterberg, Anders Jimmy et al. (2013) Optimizing heterologous protein production in the periplasm of E. coli by regulating gene expression levels. Microb Cell Fact 12:24 |
| Luirink, Joen; Yu, Zhong; Wagner, Samuel et al. (2012) Biogenesis of inner membrane proteins in Escherichia coli. Biochim Biophys Acta 1817:965-76 |
| Nørholm, Morten H H; Light, Sara; Virkki, Minttu T I et al. (2012) Manipulating the genetic code for membrane protein production: what have we learnt so far? Biochim Biophys Acta 1818:1091-6 |
| Schlegel, Susan; Lofblom, John; Lee, Chiara et al. (2012) Optimizing membrane protein overexpression in the Escherichia coli strain Lemo21(DE3). J Mol Biol 423:648-59 |
| Klepsch, M M; Kovermann, M; Low, C et al. (2011) Escherichia coli peptide binding protein OppA has a preference for positively charged peptides. J Mol Biol 414:75-85 |
| Maddalo, Gianluca; Chovanec, Peter; Stenberg-Bruzell, Filippa et al. (2011) A reference map of the membrane proteome of Enterococcus faecalis. Proteomics 11:3935-41 |
| Klepsch, Mirjam M; Persson, Jan O; de Gier, Jan-Willem L (2011) Consequences of the overexpression of a eukaryotic membrane protein, the human KDEL receptor, in Escherichia coli. J Mol Biol 407:532-42 |
| Maddalo, Gianluca; Stenberg-Bruzell, Filippa; Götzke, Hansjörg et al. (2011) Systematic analysis of native membrane protein complexes in Escherichia coli. J Proteome Res 10:1848-59 |
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