title: Overexpression and purification of human calcitonin gene-related peptide-receptor component protein in Escherichia coli.
abstract: Calcitonin gene-related peptide (CGRP) is a neuropeptide secreted by the central and peripheral nervous system nerves that has important physiological functions such as vasodilation, cardiotonic actions, metabolic and pro-inflammatory effects. The CGRP receptor is unique among G-protein coupled receptors in that a functional CGRP receptor consists of at least three proteins: calcitonin like receptor (CLR), receptor activity modifying protein (RAMP1) and receptor component protein (RCP). RCP is a required factor in CGRP-mediated signal transduction and it couples the CGRP receptor to the signal transduction pathway. Here, we describe methods to overexpress and purify RCP for structure-function studies. Human RCP was cloned and overexpressed with a poly-histidine tag and as a maltose binding protein (MBP) fusion in Escherichia coli using commercially available expression vectors. While His tagged RCP is prone to aggregation, solubility is improved when RCP is expressed as a MBP fusion. Expression and purification procedures for these constructs are described. Results from these studies will facilitate structural analysis of human RCP, and allow further understanding of RCP function.
authors.affiliation: Department of Biochemistry and Molecular Biology, Miller School of Medicine, University of Miami, P.O. Box 016129, Miami, FL 33101-6129, USA.
authors.forename: Adviye A
authors.initials: AA
authors.lastname: Tolun
authors.forename: Ian M
authors.initials: IM
authors.lastname: Dickerson
authors.forename: Arun
authors.initials: A
authors.lastname: Malhotra
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D002942:Circular Dichroism; D003001:Cloning, Molecular; D017124:Conserved Sequence; D004926:Escherichia coli / Q000235:genetics; D006801:Humans; D008969:Molecular Sequence Data; D018015:Receptors, Calcitonin Gene-Related Peptide / Q000737:chemistry / Q000235:genetics*; D011994:Recombinant Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D013045:Species Specificity
chemical_list: D018015:Receptors, Calcitonin Gene-Related Peptide; D011994:Recombinant Proteins