title: Cloning, expression, purification, and characterization of the membrane protein UncI from Escherichia coli.
abstract: The Escherichia coli unc-operon encodes the genes for the subunits of the F0F1-ATP synthase and an integral membrane protein of unknown function called UncI. UncI influences the cell-growth and activity of F0F1, but its exact function is still unknown. The expression level is too low to extract milligram amounts of UncI from E. coli membranes and the existing purification protocol based on methanol/chloroform is not suitable for structural and functional studies. Here we present protocols to increase the expression level, to purify UncI in a detergent where UncI is monodisperse, and we characterize its oligomeric state.
authors.affiliation: Center for Cellular Imaging and Nano Analytics, Biozentrum, University of Basel, CH-4058 Basel, Switzerland.
authors.forename: Claudia
authors.initials: C
authors.lastname: Hartmann
authors.forename: Andreas
authors.initials: A
authors.lastname: Engel
mesh_terms: D025181:ATP Synthetase Complexes / Q000378:metabolism; D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism; D002850:Chromatography, Gel; D003001:Cloning, Molecular; D017931:DNA Primers / Q000737:chemistry / Q000235:genetics; D004926:Escherichia coli / Q000235:genetics* / Q000378:metabolism; D015870:Gene Expression; D008565:Membrane Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism; D009876:Operon / Q000235:genetics*; D010957:Plasmids; D058105:Polymerization; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism; D014169:Transformation, Bacterial
chemical_list: D001426:Bacterial Proteins; D017931:DNA Primers; D008565:Membrane Proteins; D011994:Recombinant Proteins; D025181:ATP Synthetase Complexes