Cell-free expression for nanolipoprotein particles: Building a high-throughput membrane protein solubility platform

Jenny A. Cappuccio, Angela K. Hinz, Edward A. Kuhn, Julia E. Fletcher, Erin S. Arroyo, Paul T. Henderson, Craig D. Blanchette, Vickie L. Walsworth, Michele H. Corzett, Richard J. Law, Joseph B. Pesavento, Brent W. Segelke, Todd A. Sulchek, Brett A. Chromy, Federico Katzen, Todd Peterson, Graham Bench, Wieslaw Kudlicki, Paul D. Hoeprich, Matthew A. Coleman

Research output: Chapter in Book/Report/Conference proceedingChapter

Abstract

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.

Original languageEnglish (US)
Title of host publicationMethods in Molecular Biology
Pages273-295
Number of pages23
Volume498
DOIs
StatePublished - 2009
Externally publishedYes

Publication series

NameMethods in Molecular Biology
Volume498
ISSN (Print)10643745

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Keywords

  • Apolipoprotein
  • Bacteriorhodopsin
  • Cell-free expression
  • DMPC
  • Fluorescence
  • GPCR
  • In vitro expression
  • Lipid membranes
  • Membrane protein
  • Microarray
  • Nanodisc
  • Nanolipoprotein particles
  • NLP
  • Protein interactions
  • Protein microarray
  • RHDL

ASJC Scopus subject areas

  • Molecular Biology
  • Genetics

Cite this

Cappuccio, J. A., Hinz, A. K., Kuhn, E. A., Fletcher, J. E., Arroyo, E. S., Henderson, P. T., Blanchette, C. D., Walsworth, V. L., Corzett, M. H., Law, R. J., Pesavento, J. B., Segelke, B. W., Sulchek, T. A., Chromy, B. A., Katzen, F., Peterson, T., Bench, G., Kudlicki, W., Hoeprich, P. D., & Coleman, M. A. (2009). Cell-free expression for nanolipoprotein particles: Building a high-throughput membrane protein solubility platform. In Methods in Molecular Biology (Vol. 498, pp. 273-295). (Methods in Molecular Biology; Vol. 498). https://doi.org/10.1007/978-1-59745-196-3-18