title: Biosynthesis and purification of a hydrophobic peptide from transmembrane domains of G-protein-coupled CB2 receptor.
abstract: A major challenge for the structural study of the seven-transmembrane G-protein-coupled receptors is to obtain a sufficient amount of purified protein at the milligram level, which is required for either nuclear magnetic resonance (NMR) spectroscopy or X-ray crystallography. In order to develop a high-yield and cost-effective method, and also to obtain preliminary structural information for the computer modeling of the three-dimensional receptor structural model, a highly hydrophobic peptide from human cannabinoid subtype 2 receptor CB2(65-101), was chosen to develop high-yield membrane protein expression and purification methods. The peptide included the second transmembrane helix with the associated loop regions of the CB2 receptor. It was over-expressed in Escherichia coli, with a modified TrpDelta LE1413 (TrpLE) leading fusion sequence and a nine-histidine tag, and was then separated and purified from the tag in a preparative scale. An experimental protocol for the chemical cleavage of membrane protein fragment was developed using cyanogen bromide to remove the TrpLE tag from the hydrophobic fusion protein. In addition, protein uniformly labeled with isotopic 15N was obtained by expression in 15N-enriched minimum media. The developed and optimized preparation scheme of expression, cleavage, and purification provided a sufficient amount of peptide for NMR structure analysis and other biophysical studies that will be reported elsewhere. The process of fusion protein cleavage following purification was monitored by high-performance liquid chromatography (HPLC) and mass spectrometry (MS), and the final sample was validated by MS and circular dichroism experiments.
authors.affiliation: Department of Pharmaceutical & Pharmacological Sciences, College of Pharmacy, University of Houston, Houston, TX 77204-5037, USA.
authors.forename: H
authors.initials: H
authors.lastname: Zheng
authors.forename: J
authors.initials: J
authors.lastname: Zhao
authors.forename: S
authors.initials: S
authors.lastname: Wang
authors.forename: C-M
authors.initials: CM
authors.lastname: Lin
authors.forename: T
authors.initials: T
authors.lastname: Chen
authors.forename: D H
authors.initials: DH
authors.lastname: Jones
authors.forename: C
authors.initials: C
authors.lastname: Ma
authors.forename: S
authors.initials: S
authors.lastname: Opella
authors.forename: X-Q
authors.initials: XQ
authors.lastname: Xie
mesh_terms: D001483:Base Sequence; D006801:Humans; D057927:Hydrophobic and Hydrophilic Interactions; D008969:Molecular Sequence Data; D010455:Peptides / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification*; D017434:Protein Structure, Tertiary; D043885:Receptor, Cannabinoid, CB2 / Q000737:chemistry* / Q000235:genetics*; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000302:isolation & purification
chemical_list: D010455:Peptides; D043885:Receptor, Cannabinoid, CB2; D011993:Recombinant Fusion Proteins