Insertion of membrane proteins into discoidal membranes using a cell-free protein expression approach

Federico Katzen, Julia E. Fletcher, Jian Ping Yang, Douglas Kang, Todd C. Peterson, Jenny A. Cappuccio, Craig D. Blanchette, Todd Sulchek, Brett A. Chromy, Paul D. Hoeprich, Matthew A Coleman, Wieslaw Kudlicki

Research output: Contribution to journalArticle

92 Scopus citations

Abstract

We report a cell-free approach for expressing and inserting integral membrane proteins into water- soluble particles composed of discoidal apolipoprotein-lipid bilayers. Proteins are inserted into the particles, circumventing the need of extracting and reconstituting the product into membrane vesicles. Moreover, the planar nature of the membrane support makes the protein freely accessible from both sides of the lipid bilayer. Complexes are successfully purified by means of the apoplipoprotein component or by the carrier protein. The method significantly enhances the solubility of a variety of membrane proteins with different functional roles and topologies. Analytical assays for a subset of model membrane proteins indicate that proteins are correctly folded and active. The approach provides a platform amenable to high-throughput structural and functional characterization of a variety of traditionally intractable drug targets.

Original languageEnglish (US)
Pages (from-to)3535-3542
Number of pages8
JournalJournal of Proteome Research
Volume7
Issue number8
DOIs
StatePublished - Aug 2008
Externally publishedYes

Keywords

  • Cell-free
  • Membrane protein
  • Nanodisc
  • Protein expression
  • Proteomics

ASJC Scopus subject areas

  • Biochemistry
  • Chemistry(all)

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  • Cite this

    Katzen, F., Fletcher, J. E., Yang, J. P., Kang, D., Peterson, T. C., Cappuccio, J. A., Blanchette, C. D., Sulchek, T., Chromy, B. A., Hoeprich, P. D., Coleman, M. A., & Kudlicki, W. (2008). Insertion of membrane proteins into discoidal membranes using a cell-free protein expression approach. Journal of Proteome Research, 7(8), 3535-3542. https://doi.org/10.1021/pr800265f