title: Purification and functional reconstitution of the recombinant large mechanosensitive ion channel (MscL) of Escherichia coli.
abstract: The large mechanosensitive ion channel (MscL) of Escherichia coli was expressed on a plasmid encoding MscL as a fusion protein with glutathione S-transferase in an Escherichia coli strain containing a disruption in the chromosomal mscL gene. After purification of the fusion protein using glutathione-coated beads, thrombin cleavage allowed recovery of the MscL protein. The purified protein was reconstituted into artificial liposomes and found to be fully functional when examined with the patch-clamp technique. The reconstituted recombinant MscL protein formed ion channels that exhibited characteristic conductance and pressure sensitivity and were blocked by the mechanosensitive ion channel inhibitor gadolinium. The recombinant MscL protein was also used to raise specific anti-MscL polyclonal antibodies which abolished channel activity when preincubated with the MscL protein.
authors.affiliation: Department of Pharmacology, University of Western Australia, Nedlands.
authors.forename: C C
authors.initials: CC
authors.lastname: Häse
authors.forename: A C
authors.initials: AC
authors.lastname: Le Dain
authors.forename: B
authors.initials: B
authors.lastname: Martinac
mesh_terms: D000595:Amino Acid Sequence; D001483:Base Sequence; D004553:Electric Conductivity; D004926:Escherichia coli / Q000235:genetics / Q000502:physiology*; D029968:Escherichia coli Proteins; D005682:Gadolinium / Q000494:pharmacology; D015640:Ion Channel Gating / Q000502:physiology*; D007473:Ion Channels / Q000096:biosynthesis / Q000235:genetics / Q000276:immunology / Q000378:metabolism*; D008081:Liposomes / Q000378:metabolism; D008969:Molecular Sequence Data; D018408:Patch-Clamp Techniques; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000378:metabolism
chemical_list: D029968:Escherichia coli Proteins; D007473:Ion Channels; D008081:Liposomes; C086596:MscL protein, E coli; D011994:Recombinant Proteins; D005682:Gadolinium