Pore-forming proteins are becoming increasingly important for different
biomedical and nanotechnological applications. Different classes of pore
forming proteins require diverse isolation and purification strategies,
which often include detergent solubilisation or refolding steps.
Therefore a rapid functional characterisation of the purified protein
species is of great relevance. A direct method to assess the
functionality of pore forming proteins is to measure an ionic current
through a protein pore embedded in an artificial lipid bilayer. Here we
report on the design of a setup for parallel high-resolution
electrophysiological recordings allowing for the screening of functional
activity of different pore-forming proteins in controlled lipid
environments on the single-molecule level.
The developed recording chamber is based on a SU-8 coated printed
circuit board containing 4 cavities (50µm) with individual
microelectrodes (Micro Electrode Cavity Array (MECA) as well as a common
ground electrode. 4 suspended lipid bilayers can be self-assembled on
the nonpolar chip surface from phospholipids in organic solvent. The
MECA-PCB is connected to a multichannel amplifier capable of
simultaneous recording of electrical activity from the ion channels
functionally reconstituted into the bilayers with high-bandwidth and low
noise (<1 pA rms @ 10 kHz).
The capability of the MECA technology for the protein functionality
screening has been tested and validated for a wide variety of pore
forming proteins including antimicrobial peptides, soluble bacterial
toxins, bacterial outer membrane proteins, viroporins and ion channels.
The combination of the cell-free protein expression system and
MECA-PCB-chip electrophysiology allowed for functional characterization
of the synthesized channels within several hours starting from the DNA
template. The current PCB-chip design can be easily adapted for higher
throughput and automated lipid bilayer formation as recently reported1.