Cell-free co-expression of functional membrane proteins and apolipoprotein, forming soluble nanolipoprotein particles

Jenny A. Cappuccio, Craig D. Blanchette, Todd A. Sulchek, Erin S. Arroyo, Joel M. Kralj, Angela K. Hinz, Edward A. Kuhn, Brett A. Chromy, Brent W. Segelke, Kenneth J. Rothschild, Julia E. Fletcher, Federico Katzen, Todd C. Peterson, Wieslaw A. Kudlicki, Graham Bench, Paul D. Hoeprich, Matthew A Coleman

Research output: Contribution to journalArticle

75 Scopus citations

Abstract

Here we demonstrate rapid production of solubilized and functional membrane protein by simultaneous cell-free expression of an apolipoprotein and a membrane protein in the presence of lipids, leading to the self-assembly of membrane protein-containing nanolipoprotein particles (NLPs). NLPs have shown great promise as a biotechnology platform for solubilizing and characterizing membrane proteins. However, current approaches are limited because they require extensive efforts to express, purify, and solubilize the membrane protein prior to insertion into NLPs. By the simple addition of a few constituents to cell-free extracts, we can produce membrane proteins in NLPs with considerably less effort. For this approach an integral membrane protein and an apolipoprotein scaffold are encoded by two DNA plasmids introduced into cell-free extracts along with lipids. For this study reported here we used plasmids encoding the bacteriorhodopsin (bR) membrane apoprotein and scaffold protein δ1-49 apolipoprotein A-1 fragment (δ49A1). Cell free co-expression of the proteins encoded by these plasmids, in the presence of the cofactor all-trans-retinal and dimyristoylphosphatidylcholine, resulted in production of functional bR as demonstrated by a 5-nm shift in the absorption spectra upon light adaptation and characteristic time-resolved FT infrared difference spectra for the bR → M transition. Importantly the functional bR was solubilized in discoidal bR-NLPs as determined by atomic force microscopy. A survey study of other membrane proteins co-expressed with δ49A1 scaffold protein also showed significantly increased solubility of all of the membrane proteins, indicating that this approach may provide a general method for expressing membrane proteins enabling further studies.

Original languageEnglish (US)
Pages (from-to)2246-2253
Number of pages8
JournalMolecular and Cellular Proteomics
Volume7
Issue number11
DOIs
StatePublished - Nov 2008
Externally publishedYes

ASJC Scopus subject areas

  • Biochemistry
  • Molecular Biology
  • Analytical Chemistry

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    Cappuccio, J. A., Blanchette, C. D., Sulchek, T. A., Arroyo, E. S., Kralj, J. M., Hinz, A. K., Kuhn, E. A., Chromy, B. A., Segelke, B. W., Rothschild, K. J., Fletcher, J. E., Katzen, F., Peterson, T. C., Kudlicki, W. A., Bench, G., Hoeprich, P. D., & Coleman, M. A. (2008). Cell-free co-expression of functional membrane proteins and apolipoprotein, forming soluble nanolipoprotein particles. Molecular and Cellular Proteomics, 7(11), 2246-2253. https://doi.org/10.1074/mcp.M800191-MCP200