Biosynthesis, purification, and characterization of a cannabinoid receptor 2 fragment (CB2(271-326)).
Obtaining sufficient amount of purified G-protein coupled receptors (GPCRs) is almost always one of the major challenges for their structural studies. CB2(271-326), a human cannabinoid receptor 2 (CB2) fragment comprising part of the third extracellular loop (EL3), the seventh transmembrane domain (TM7) and C-terminal juxtamembrane region of the receptor, was over-expressed as a fusion protein into inclusion body (IB) of Escherichia coli. The fusion protein was purified by histidine-selected nickel affinity chromatography under denaturing conditions. Then, the fusion protein IBs were solubilized in detergent (Brij58) and the expression fusion leader sequence (TrpLE) was specifically cleaved with tobacco etch virus (TEV) protease. The target fragment, CB2(271-326), was subsequently purified by reverse-phase HPLC and confirmed by SDS-PAGE and mass spectrometry. This hydrophobic fragment can refold in mild detergents digitonin and Brij58. Circular dichroism (CD) spectroscopy of CB2(271-326) in digitonin and Brij58 micelles showed that the fragment adopts a more than 75% alpha-helical structure, with the remainder having beta-strand structure. Fluorescence spectroscopy and quenching studies suggested that the C-terminal region lies near the surface of the digitonin micelles and the TM7 region is folded relatively close to the center of the micelles. This study may provide an alternative strategy for the production and structure/functional studies of GPCRs such as CB2 receptor protein produced in the form of IBs.
Zhang
Yuxun
Y
Department of Pharmaceutical Sciences, School of Pharmacy, University of Pittsburgh, 10016 BST3, 3501 Fifth Avenue, Pittsburgh, PA 15260, USA.
Xie
Xiang-Qun
XQ
D020816:Amino Acid Motifs; D002592:Cetomacrogol / Q000737:chemistry; D002846:Chromatography, Affinity; D002851:Chromatography, High Pressure Liquid; D002942:Circular Dichroism; D003902:Detergents / Q000737:chemistry; D004072:Digitonin / Q000737:chemistry; D004591:Electrophoresis, Polyacrylamide Gel; D010450:Endopeptidases / Q000737:chemistry; D004926:Escherichia coli / Q000235:genetics; D006801:Humans; D002479:Inclusion Bodies / Q000737:chemistry / Q000378:metabolism; D009532:Nickel / Q000737:chemistry; D010446:Peptide Fragments / Q000096:biosynthesis* / Q000737:chemistry / Q000302:isolation & purification*; D011489:Protein Denaturation; D017510:Protein Folding; D021382:Protein Sorting Signals; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D043885:Receptor, Cannabinoid, CB2 / Q000096:biosynthesis* / Q000737:chemistry / Q000302:isolation & purification*; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis* / Q000737:chemistry / Q000302:isolation & purification*; D013050:Spectrometry, Fluorescence
D003902:Detergents; D010446:Peptide Fragments; D021382:Protein Sorting Signals; D043885:Receptor, Cannabinoid, CB2; D011993:Recombinant Fusion Proteins; D009532:Nickel; D002592:Cetomacrogol; D010450:Endopeptidases; C099488:TEV protease; D004072:Digitonin