Summary
Membrane-associated proteins and protein complexes account for approximately a third or more of the proteins in the cell (1, 2). These complexes mediate essential cellular processes; including signal transduc-tion, transport, recognition, bioenergetics and cell–cell communication. In general, membrane proteins are challenging to study because of their insolubility and tendency to aggregate when removed from their protein lipid bilayer environment. This chapter is focused on describing a novel method for producing and solubilizing membrane proteins that can be easily adapted to high-throughput expression screening. This process is based on cell-free transcription and translation technology coupled with nanolipoprotein par ticles (NLPs), which are lipid bilayers confined within a ring of amphipathic protein of defined diameter. The NLPs act as a platform for inserting, solubilizing and characterizing functional membrane proteins. NLP component proteins (apolipoproteins), as well as membrane proteins can be produced by either traditional cell-based or as discussed here, cell-free expression methodologies.
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Acknowledgments
This work performed under the auspices of the U.S. Department of Energy by Lawrence Livermore National Laboratory under Contract DE-AC52—07NA27344, with support from the Labora tory Directed Research and Development Office (LDRD) 06-SI-003 awarded to PDH. UCRL-BOOK-235838. The authors are grateful to Drs. Karl Weisgraber and Robert Ryan for helpful discussions and providing reagents.
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Cappuccio, J.A. et al. (2009). Cell-Free Expression for Nanolipoprotein Particles: Building a High-Throughput Membrane Protein Solubility Platform. In: Doyle, S.A. (eds) High Throughput Protein Expression and Purification. Methods in Molecular Biology, vol 498. Humana Press. https://doi.org/10.1007/978-1-59745-196-3_18
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DOI: https://doi.org/10.1007/978-1-59745-196-3_18
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