Biosynthesis, purification, and characterization of a cannabinoid receptor 2 fragment (CB2271–326)

Abstract

Obtaining sufficient amount of purified G-protein coupled receptors (GPCRs) is almost always one of the major challenges for their structural studies. CB2 , 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 histidineselected 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 , 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 in digitonin and Brij58 micelles showed that the fragment adopts a more than 75% α-helical structure, with the remainder having β-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. KeywordsCannabinoid receptor 2; transmembrane domain; inclusion body; affinity chromatography; HPLC; circular dichroism; fluorescence Since the discovery of the CB2 receptor in 1993 [1], there has been a growing interest to clarify the importance of this GPCR membrane protein in human physiology, and to investigate it as a possible target for current and future drug development. The CB2 receptor seems to have a significant role in mediating the immunological functions of cannabinoid receptor ligands. Thus, the CB2 receptor is a promising target for drug development that aims at management of neurological pain, inflammation, osteoporosis, and growth of malignant gliomas and tumors * Corresponding author. Fax: +1 412 383 5276. E-mail address: xix15@pitt.edu (X-Q. Xie). Publisher's Disclaimer: This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. [3][4][5][6][7]. In general, the Escherichia coli expression system is the most attractive strategy due to its advantages of simplicity of use, low cost, easy scale-up, and homogeneous protein production. Though few attempts were promising to express full length CB2 receptor in ...

Advances in Methods to Characterize Ligand-Induced Ionic Lock and Rotamer Toggle Molecular Switch in G Protein-Coupled Receptors

“…Although Escherichia coli has obvious advantages of fast and low-cost production of GPCR and/or GPCR fragments, receptor may not be translocated to the membrane and hence render nonfunctional. Substantial success has been achieved for the production of the GPCR fragments in the E. coli (Chowdhury et al, 2012; Xie, Zheng, & Zhao, 2004; Zhang & Xie, 2008; Zheng et al, 2005). Expression and posttranslational modification of recombinant GPCR is similar to that of mammalian cells, and GPCR produced are almost identical in insect and mammalian cells except for some minor differences.…”

Preparation of stable isotope-labeled peripheral cannabinoid receptor CB2 by bacterial fermentation

“…Expression in Escherichia coli ( E. coli ) cells [14] is characterized by high growth rates [13], significant biomass yields, and has a potential for producing homogenous (albeit non-glycosylated) [13, 14] target proteins. E. coli cells can grow in a minimal medium of a defined composition which simplifies procedures for the incorporation of stable isotope-labeled precursors into the recombinant receptor [13].…”

Mistic and TarCF as fusion protein partners for functional expression of the cannabinoid receptor 2 in Escherichia coli

“…Previously, E. coli was used in our lab to express CB2 receptor fragments by directing the fragment expression to inclusion bodies using the Trp LE leader sequence [31–32]. The CB2 receptor fragment produced in E. coli and reconstituted in Brij 58 showed > 75% preservation of the alpha helical structure [33]. However, the methodology developed in these studies may not be applied to the intact receptor without substantial modifications.…”