Chimeric bacterial-human NaV17 sodium channels expressed in E coli
Powl, Andrew M. and Wallace, Bonnie A. (2009) Chimeric bacterial-human NaV17 sodium channels expressed in E coli. Biophysical Journal 96 (3(S1)), 13a-13a. ISSN 0006-3495.
Voltage-gated sodium channels selectively transport sodium ions across cellular membranes in response to changes in membrane potential. Prokaryotic voltage-gated sodium channels are homotetramers, each monomer containing six transmembrane helices (S1–S6), consisting of a voltage-sensing subdomain (S1–S4) and a pore-forming subdomain (S5–S6). In eukaryotes, sodium channels consist of a single polypeptide chain containing four similar domains, each with six transmembrane helices (S1–S6), which create pseudo-tetrameric channels. In humans, genetic diseases associated with the NaV1.7 sodium channel isoform include loss-of-function (i.e. channelopathy-associated indifference to pain), in addition to gain-of-function inherited painful neuropathies; hence, this channel is an important target for drug discovery. Expression of eukaryotic membrane proteins in E. coli is often a difficult task, resulting in cell death, no expression of the target protein, or proteins inserted into inclusion bodies. In order to enable the expression of crucial functional regions of eukaryotic sodium channels, we have developed a method for creating chimeric proteins with the N-terminal subdomain of a prokaryotic homologue, and the C-terminal subdomain of the eukaryotic protein of interest, thereby tricking the bacterial host into expressing a protein with functional regions of interest from the eukaryote. In this study we designed, constructed, expressed, and characterised a number of sodium channel chimeras containing the voltage sensor (S1–S4) from B. halodurans NaChBac and the pore regions (S5–S6) from domains II and III of human NaV1.7, including the S4–S5 linkers from either the bacterial or eukaryotic protein.
|School:||Birkbeck Schools and Departments > School of Science > Biological Sciences|
|Research Centre:||Bioinformatics, Bloomsbury Centre for, Structural Molecular Biology, Institute of (ISMB)|
|Date Deposited:||04 Aug 2010 14:09|
|Last Modified:||07 Dec 2016 15:05|
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