title: Affinity Capturing and Surface Enrichment of a Membrane Protein Embedded in a Continuous Supported Lipid Bilayer.
abstract: Investigations of ligand-binding kinetics to membrane proteins are hampered by their poor stability and low expression levels, which often translates into sensitivity-related limitations impaired by low signal-to-noise ratios. Inspired by affinity capturing of water-soluble proteins, which utilizes water as the mobile phase, we demonstrate affinity capturing and local enrichment of membrane proteins by using a fluid lipid bilayer as the mobile phase. Specific membrane-protein capturing and enrichment in a microfluidic channel was accomplished by immobilizing a synthesized trivalent nitrilotriacetic acid (tris-NTA)-biotin conjugate. A polymer-supported lipid bilayer containing His6-tagged β-secretase (BACE) was subsequently laterally moved over the capture region by using a hydrodynamic flow. Specific enrichment of His6-BACE in the Ni2+-NTA-modified region of the substrate resulted in a stationary three-fold increase in surface coverage, and an accompanied increase in ligand-binding response.
authors: Discovery Sciences AstraZeneca R&D Mölndal 43183 Mölndal Sweden.
authors: Anders
authors: A
authors: Gunnarsson
authors: Department of Applied Physics Chalmers University of Technology 412 96 Göteborg Sweden.
authors: Lisa
authors: L
authors: Simonsson Nyström
authors: Department of Applied Physics Chalmers University of Technology 412 96 Göteborg Sweden.
authors: Sabina
authors: S
authors: Burazerovic
authors: Discovery Sciences AstraZeneca R&D Mölndal 43183 Mölndal Sweden.
authors: Jenny
authors: J
authors: Gunnarsson
authors: Discovery Sciences AstraZeneca R&D Mölndal 43183 Mölndal Sweden.
authors: Arjan
authors: A
authors: Snijder
authors: Discovery Sciences AstraZeneca R&D Mölndal 43183 Mölndal Sweden.
authors: Stefan
authors: S
authors: Geschwindner
authors: Department of Applied Physics Chalmers University of Technology 412 96 Göteborg Sweden.
authors: Fredrik
authors: F
authors: Höök
keywords: His-tag; affinity purification; membrane proteins; supported lipid bilayer; surface chemistry