title: Bacterial expression and purification of a heterodimeric adrenomedullin receptor extracellular domain complex using DsbC-assisted disulfide shuffling.
abstract: Adrenomedullin (AM) is a peptide hormone that is a potent vasodilator and is essential for vascular development. The AM receptor is a heterodimeric cell surface receptor composed of the calcitonin receptor-like receptor (CLR), a class B G protein-coupled receptor, in association with either of two receptor activity modifying protein (RAMP) coreceptors, RAMP2 or -3. The extracellular domains (ECDs) of CLR and the RAMPs form the primary AM binding site. Here, we present novel methodology for expression and purification of a heterodimeric AM receptor ECD complex as an MBP-CLR ECD fusion protein in association with the RAMP2 ECD. Co-expression of the RAMP2 ECD with the disulfide bond isomerase DsbC in the oxidizing cytoplasm of E. coli trxB gor enabled proper disulfide formation in vivo. The isolated RAMP2 ECD was purified to homogeneity. Co-expression of a soluble MBP-CLR ECD fusion protein with DsbC in E. coli trxB gor yielded a heterogeneous mixture of species with misfolded ECD. Incubation of affinity-purified MBP-CLR ECD in vitro with purified RAMP2 ECD, DsbC, and glutathione redox buffer promoted proper folding of the CLR ECD and formation of a stable MBP-CLR ECD:RAMP2 ECD complex that was purified by size-exclusion chromatography and which exhibited specific AM binding. Approximately 40mg of highly purified complex was obtained starting with 6L bacterial cultures for each protein. The methodology reported here will facilitate structure/function studies of the AM receptor.
authors.affiliation: Department of Biochemistry and Molecular Biology, University of Oklahoma Health Sciences Center, 975 N.E. 10th Street, Oklahoma City, OK 73104, USA.
authors.forename: Heather E
authors.initials: HE
authors.lastname: Hill
authors.forename: Augen A
authors.initials: AA
authors.lastname: Pioszak
mesh_terms: D000595:Amino Acid Sequence; D001665:Binding Sites; D058286:Calcitonin Receptor-Like Protein / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D004220:Disulfides / Q000737:chemistry; D006801:Humans; D046912:Multiprotein Complexes / Q000737:chemistry / Q000302:isolation & purification*; D011485:Protein Binding; D017434:Protein Structure, Tertiary / Q000235:genetics; D058262:Receptor Activity-Modifying Protein 1; D058263:Receptor Activity-Modifying Protein 2 / Q000737:chemistry / Q000302:isolation & purification; D058264:Receptor Activity-Modifying Protein 3 / Q000737:chemistry / Q000302:isolation & purification; D058265:Receptors, Adrenomedullin / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification*; D015398:Signal Transduction; D014665:Vasodilator Agents / Q000737:chemistry / Q000302:isolation & purification
chemical_list: D058286:Calcitonin Receptor-Like Protein; D004220:Disulfides; D046912:Multiprotein Complexes; D058262:Receptor Activity-Modifying Protein 1; D058263:Receptor Activity-Modifying Protein 2; D058264:Receptor Activity-Modifying Protein 3; D058265:Receptors, Adrenomedullin; D014665:Vasodilator Agents