A functional approach to molecular identification of the large conductance mechanosensitive channel (MscL) from Escherichia coli cell envelope is described. Isolated membranes were solubilized in octylglucoside, and proteins were fractionated on several columns. Individual or pools of fractions were reconstituted into azolectin liposomes, which were then examined for the characteristic MscL activity (2.5-nS mechnoactivated currents) by the patch-clamp technique. Averaging the numbers of MscL conducting units in several patches allowed us to rule out inactive fractions and select MscL-containing fractions for subsequent steps of enrichment. We have thereby correlated MscL activity with the presence of a 17-kDa protein, whose gene, mscL, was then cloned. We confirmed a direct relationship between the mscL product and the channel activity by heterologous expression of this gene in two different systems. Here we describe the techniques used to record the characteristic MscL currents from liposomes reconstituted with material from MscL-expressing yeasts or with canine microsomes into which MscL protein was synthesized by a cell-free expression system.
ASJC Scopus subject areas
- Molecular Biology
- Biochemistry, Genetics and Molecular Biology(all)