1 research outputs found
Preparation of Tethered-Lipid Bilayers on Gold Surfaces for the Incorporation of Integral Membrane Proteins Synthesized by Cell-Free Expression
There
is an increasing interest to express and study membrane proteins
in vitro. New techniques to produce and insert functional membrane
proteins into planar lipid bilayers have to be developed. In this
work, we produce a tethered lipid bilayer membrane (tBLM) to provide
sufficient space for the incorporation of the integral membrane protein
(IMP) Aquaporin Z (AqpZ) between the tBLM and the surface of the sensor.
We use a gold (Au)-coated sensor surface compatible with mechanical
sensing using a quartz crystal microbalance with dissipation monitoring
(QCM-D) or optical sensing using the surface plasmon resonance (SPR)
method. tBLM is produced by vesicle fusion onto a thin gold film,
using phospholipid-polyethylene glycol (PEG) as a spacer. Lipid vesicles
are composed of 1-palmitoyl-2-oleoyl-<i>sn</i>-glycero-3-phosphocholine
(POPC) and 1,2-distearoyl-<i>sn</i>-glycero-3-phosphoethanolamine-<i>N</i>-poly(ethyleneglycol)-2000-<i>N</i>-[3-(2-pyridyldithio)propionate],
so-called DSPE-PEG-PDP, at different molar ratios (respectively, 99.5/0.5,
97.5/2.5, and 95/5 mol %), and tBLM formation is characterized using
QCM-D, SPR, and atomic force technology (AFM). We demonstrate that
tBLM can be produced on the gold surface after rupture of the vesicles
using an α helical (AH) peptide, derived from hepatitis C virus
NS5A protein, to assist the fusion process. A cell-free expression
system producing the E. coli integral
membrane protein Aquaporin Z (AqpZ) is directly incubated onto the
tBLMs for expression and insertion of the IMP at the upper side of
tBLMs. The incorporation of AqpZ into bilayers is monitored by QCM-D
and compared to a control experiment (without plasmid in the cell-free
expression system). We demonstrate that an IMP such as AqpZ, produced
by a cell-free expression system without any protein purification,
can be incorporated into an engineered tBLM preassembled at the surface
of a gold-coated sensor