Direct solubilization of heterologously expressed membrane proteins by incorporation into nanoscale lipid bilayers

Natanya R. Civjan, Timothy H. Bayburt, Mary A Schuler, Stephen Sligar

Research output: Contribution to journalArticle

Abstract

One of the biggest challenges in the field of proteomics is obtaining functional membrane proteins solubilized and dispersed into a physiologically relevant environment that maintains the spectrum of in vivo activities. Here we describe a system composed of nanoscale self-assembled particles, termed Nanodiscs™, which contain a single phospholipid bilayer stabilized by ah an encircling membrane scaffold protein (MSP). Using microsomal membranes of baculovirus-infected Spodoptera frugiperda (Sf9) insect cells overexpressing an N-terminally anchored cytochrome P450 monooxygenase (P450), we demonstrate that target membrane proteins can be directly solubilized and incorporated into distinct populations of Nanodiscs, which can be separated by size chromatography. We show that formation of these Nanodiscs from insect cell membranes allows for the compartmentalization into soluble nanostructures that provide a natural membrane bilayer that avoids the aggregation of membrane proteins often encountered in other reconstitution procedures. Lipid composition analysis and substrate binding analysis of size-fractionated Nanodiscs arrayed in microtiter plates further demonstrates that the Nanodisc system effectively disperses the overexpressed membrane protein into monodispersed bilayers containing biochemically defined lipid components and the target protein in its native form suitable for sensitive high-throughput substrate binding analysis.

Original languageEnglish (US)
Pages (from-to)556-563
Number of pages8
JournalBioTechniques
Volume35
Issue number3
StatePublished - Sep 1 2003

ASJC Scopus subject areas

  • Biotechnology
  • Biochemistry, Genetics and Molecular Biology(all)

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