Addressable adsorption of lipid vesicles and subsequent protein interaction studies
Artikel i vetenskaplig tidskrift, 2008

We demonstrate a convenient chip platform for the addressable immobilization of protein-loaded vesicles on a microarray for parallelized, high-throughput analysis of lipid-protein systems. Self-sorting of the vesicles on the microarray was achieved through DNA bar coding of the vesicles and their hybridization to complementary strands, which are preimmobilized in defined array positions on the chip. Imaging surface plasmon resonance in ellipsometric mode was used to monitor vesicle immobilization, protein tethering, protein-protein interactions, and chip regeneration. The immobilization strategy proved highly specific and stable and presents a mild method for the anchoring of vesicles to predefined areas of a surface, while unspecific adsorption to both noncomplementary regions and background areas is nonexistent or, alternatively, undetectable. Furthermore, histidine-tagged receptors have been stably and functionally immobilized via bis-nitrilotriacetic acid chelators already present in the vesicle membranes. It was discovered though that online loading of proteins to immobilized vesicles leads to cross contamination of previously loaded vesicles and that it was necessary to load the vesicles offline in order to obtain pure protein populations on the vesicles. We have used this cross-binding effect to our benefit by coimmobilizing two receptor subunits in different ratios on the vesicle surface and successfully demonstrated ternary complex formation with their ligand. This approach is suitable for mechanistic studies of complex multicomponent analyses involving membrane-bound systems.

I

DNA

SURFACE-PLASMON RESONANCE

IMMOBILIZATION

molecular

surface plasmon resonance

INTERFERON-RECEPTOR

biomembranes

BINDING INTERFACE

MEMBRANES

lipid bilayers

biochemistry

HISTIDINE-TAGGED PROTEINS

MICROARRAYS

ARRAYS

MATRIX

adsorption

biophysics

DNA

proteins

Författare

G. Klenkar

Chalmers University of Technology

Linköpings universitet

Björn Brian

Linköpings universitet

T. Ederth

Linköpings universitet

Gudrun Stengel

Chalmers University of Technology

Fredrik Höök

Chalmers, Teknisk fysik, Biologisk fysik

J. Piehler

Johann Wolfgang Goethe Universität Frankfurt am Main

B. Liedberg

Linköpings universitet

Biointerphases

1559-4106 (ISSN) 1934-8630 (eISSN)

Vol. 3 29-37

Ämneskategorier

Annan teknik

DOI

10.1116/1.2921867