Direct transfer of membrane proteins from bacteria to planar bilayers for rapid screening by single-channel recording.
Although the examination of membrane proteins in planar bilayers is a powerful methodology for evaluating their pharmacology and physiological roles, introducing membrane proteins into bilayers is often a difficult process. Here, we use a mechanical probe to transfer membrane proteins directly from Escherichia coli expression colonies to artificial lipid bilayers. In this way, single-channel electrical recordings can be made from both of the major classes of membrane proteins, alpha-helix bundles and beta barrels, which are represented respectively by a K(+) channel and a bacterial pore-forming toxin. Further, we examined the bicomponent toxin leukocidin (Luk), which is composed of LukF and LukS subunits. We mixed separate LukF- and LukS-expressing colonies and transferred the mixture to a planar bilayer, which generated functional Luk pores. By this means, we rapidly screened binary combinations of mutant Luk subunits for a specific function: the ability to bind a molecular adaptor. We suggest that direct transfer from cells to bilayers will be useful in several aspects of membrane proteomics and in the construction of sensor arrays.
Holden
Matthew A
MA
Department of Chemistry, University of Oxford, Chemistry Research Laboratory, Mansfield Road, Oxford OX1 3TA, UK. matthew.holden@chem.ox.ac.uk
Jayasinghe
Lakmal
L
Daltrop
Oliver
O
Mason
Amy
A
Bayley
Hagan
H