Affinity Purification and Single-Molecule Analysis of Integral Membrane Proteins from Crude Cell-Membrane Preparations
Journal article, 2018

The function of integral membrane proteins is critically dependent on their naturally surrounding lipid membrane. Detergent-solubilized and purified membrane proteins are therefore often reconstituted into cell-membrane mimics and analyzed for their function with single-molecule microscopy. Expansion of this approach toward a broad range of pharmaceutically interesting drug targets and biomarkers however remains hampered by the fact that these proteins have low expression levels, and that detergent solubilization and reconstitution often cause protein conformational changes and loss of membrane-specific cofactors, which may impair protein function. To overcome this limitation, we here demonstrate how antibody-modified nanoparticles can be used to achieve affinity purification and enrichment of selected integral membrane proteins directly from cell membrane preparations. Nanoparticles were first bound to the ectodomain of β-secretase 1 (BACE1) contained in cell-derived membrane vesicles. In a subsequent step, these were merged into a continuous supported membrane in a microfluidic channel. Through the extended nanoparticle tag, a weak (∼fN) hydrodynamic force could be applied, inducing directed in-membrane movement of targeted BACE1 exclusively. This enabled selective thousand-fold enrichment of the targeted membrane protein while preserving a natural lipid environment. In addition, nanoparticle-targeting also enabled simultaneous tracking analysis of each individual manipulated protein, revealing how their mobility changed when moved from one lipid environment to another. We therefore believe this approach will be particularly useful for separation in-line with single-molecule analysis, eventually opening up for membrane-protein sorting devices analogous to fluorescence-activated cell sorting.

functionalized nanoparticles

single-molecule analysis

single-particle tracking

BACE-1

Membrane-protein purification

membrane vesicles

Author

Anders Lundgren

University of Natural Resources and Life Sciences, Vienna

Chalmers, Physics, Biological Physics

Björn Johansson Fast

Chalmers, Physics, Biological Physics

Stephan Block

Chalmers, Physics, Biological Physics

Björn Agnarsson

Chalmers, Physics, Biological Physics

E. Reimhult

University of Natural Resources and Life Sciences, Vienna

Anders Gunnarsson

AstraZeneca AB

Fredrik Höök

Chalmers, Physics, Biological Physics

Nano Letters

1530-6984 (ISSN) 1530-6992 (eISSN)

Vol. 18 1 381-385

Subject Categories

Biochemistry and Molecular Biology

Biophysics

Medical Biotechnology (with a focus on Cell Biology (including Stem Cell Biology), Molecular Biology, Microbiology, Biochemistry or Biopharmacy)

DOI

10.1021/acs.nanolett.7b04227

More information

Latest update

9/18/2018