title: Purification of a Tat leader peptide by co-expression with its chaperone.
abstract: We present a method for the purification of the 45 residue long leader peptide of Escherichia coli dimethyl sulfoxide reductase subunit A (DmsA(L)), a substrate of the twin arginine translocase, by co-expressing the leader peptide with its specific chaperone protein, DmsD. The peptide can be isolated from the soluble DmsA(L)/DmsD complex or conveniently from the lysate pellet fraction. The recombinant leader peptide is functionally intact as the peptide/chaperone complex can be reconstituted from purified DmsA(L) and DmsD. A construct with DmsA(L) fused to the N-terminus of DmsD (DmsA(L)-DmsD fusion) was created to further explore the properties of the leader peptide-chaperone interactions. Analytical size-exclusion chromatography in-line with multi-angle light scattering reveals that the DmsA(L)-DmsD fusion construct forms a dimer wherein each protomer binds the neighboring leader peptide. A model of this homodimeric interaction is presented.
authors.affiliation: Department of Molecular Biology and Biochemistry, Simon Fraser University, South Science Building, 8888 University Drive, Burnaby, British Columbia, Canada V5A 1S6.
authors.forename: Charles M
authors.initials: CM
authors.lastname: Stevens
authors.forename: Mark
authors.initials: M
authors.lastname: Paetzel
mesh_terms: D000595:Amino Acid Sequence; D001483:Base Sequence; D002352:Carrier Proteins / Q000096:biosynthesis* / Q000737:chemistry / Q000235:genetics; D002850:Chromatography, Gel; D029968:Escherichia coli Proteins / Q000096:biosynthesis* / Q000737:chemistry / Q000235:genetics; D006639:Histidine / Q000737:chemistry / Q000235:genetics; D047908:Intracellular Signaling Peptides and Proteins; D007506:Iron-Sulfur Proteins / Q000096:biosynthesis* / Q000737:chemistry / Q000235:genetics; D008027:Light; D008958:Models, Molecular; D018832:Molecular Chaperones / Q000096:biosynthesis* / Q000737:chemistry / Q000235:genetics; D008969:Molecular Sequence Data; D046912:Multiprotein Complexes; D009842:Oligopeptides / Q000737:chemistry / Q000235:genetics; D010088:Oxidoreductases / Q000096:biosynthesis* / Q000737:chemistry / Q000235:genetics; D011485:Protein Binding; D021382:Protein Sorting Signals; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism; D012542:Scattering, Radiation
chemical_list: D002352:Carrier Proteins; C434268:DmsD protein, E coli; D029968:Escherichia coli Proteins; C471213:His-His-His-His-His-His; D047908:Intracellular Signaling Peptides and Proteins; D007506:Iron-Sulfur Proteins; D018832:Molecular Chaperones; D046912:Multiprotein Complexes; D009842:Oligopeptides; D021382:Protein Sorting Signals; D011993:Recombinant Fusion Proteins; D006639:Histidine; D010088:Oxidoreductases; C045664:dimethyl sulfoxide reductase