Overexpression and purification of human calcitonin gene-related peptide-receptor component protein in Escherichia coli.
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.
Tolun
Adviye A
AA
Department of Biochemistry and Molecular Biology, Miller School of Medicine, University of Miami, P.O. Box 016129, Miami, FL 33101-6129, USA.
Dickerson
Ian M
IM
Malhotra
Arun
A
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
D018015:Receptors, Calcitonin Gene-Related Peptide; D011994:Recombinant Proteins