Journal
ENGINEERING IN LIFE SCIENCES
Volume 15, Issue 1, Pages 39-50Publisher
WILEY-BLACKWELL
DOI: 10.1002/elsc.201400187
Keywords
Automation; Detergent screening; High throughput; Membrane proteins
Categories
Funding
- Netherlands Organization for Scientific Research (ZON-MW AMR program)
- FRISBI [ANR-10-INSB-05-02]
- GRAL within the Grenoble Partnership for Structural Biology (PSB) [ANR-10-LABX-49-01]
Ask authors/readers for more resources
Membrane proteins constitute about one third of proteins encoded by all genomes, but only a small percentage have their structures deposited in the Protein Data Bank. One bottleneck in the pipeline from expression to structure determination is the identification of detergents that maintain the protein in a soluble, stable, and active state. Here, we describe a small-scale automated procedure to easily and rapidly screen detergents for the solubilization and purification of membrane proteins, to perform detergent exchange, or to identify conditions preserving protein interactions in complexes. Hundreds of conditions can be tested in a few hours to select detergents that keep proteins folded and nonaggregated, from single membrane preparations of cells overexpressing the protein(s) of interest. Thirty-one prokaryotic, eukaryotic, and viral membrane proteins were analyzed by our small-scale procedure to identify the best-associated detergents. Examples of results obtained with a bitopic and multitopic membrane proteins and membrane protein complexes are presented in more detail. DDM, DM, DMNG, TritonX-100, LAPAO, and Fos-12 appeared effective for successful membrane solubilization and protein purification of most selected targets. Eukaryotic proteins are in general more difficult to extract and purify from Escherichia coli membranes than prokaryotic proteins. The protocol has been developed for His-tagged proteins, but can readily be adapted to other affinity tags by adjusting the chromatography resin and the buffer composition.
Authors
I am an author on this paper
Click your name to claim this paper and add it to your profile.
Reviews
Recommended
No Data Available