Biosynthesis, Purification and Characterization of A Cannabinoid Receptor 2 Fragment (CB2271-326)

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 of immune origin, as well as immunological disorders such as multiple sclerosis [2]. However, structure-function studies and structure-based ligand design have been impeded by the absence of an experimental three-dimensional (3D) structure of the CB2 receptor. Seven transmembrane α-helical domains and the related high hydrophobicity are the major contributing factors that hinder the 3D structural study of CB2 and other GPCRs. Because of its very limited quantities obtainable from natural sources, attempts have been made to heterologously over-express CB2 [3-7]. 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 E. coli [3-5], it is still problematic to obtain the entire CB2 tertiary structure due to the difficulties to get enough yield of CB2 protein, to purify it, to entice it to fold properly, and to analyze it by NMR. Based on the above fact, we hypothesized that an alternative strategy, production of large CB2 fragments including up to two TM segments connected by a loop through over-expression in bacteria as fusion proteins, would be tractable. Structural and even functional analysis of bacterially synthesized protein fragments has become a powerful tool in the study of GPCRs and other membrane proteins [8-11]. Our lab has previously over-expressed, purified and structurally characterized CB2 fragments, including CB2180-233, CB265-101, and CB227-101, which correspond to TM1-IL1-TM2 (from the first transmembrane domain to the second intracellular loop and the second transmembrane domain), TM2 and TM5-IL3 [12-15] respectively. In these publications, the CB2 fragments were expressed as fusion proteins with a modified TrpΔLE1413 (TrpLE) leader sequence and a 9-histidine tag at the N-terminus. The TrpLE leader sequence facilitates the fusion protein to be highly expressed as IBs, which have the advantages of lower toxicity and higher yield than the strategy of insertion of the protein into the bacterial membrane. The formation of IBs also has an advantage of protecting the recombinant protein from degradation by cytosolic bacterial proteases. Also, the insolubility of IB proteins allows for removal of soluble and membrane proteins by simple centrifugation. The resulting fusion proteins were further purified by immobilized metal-ion affinity chromatography (IMAC) under denaturing conditions (6 M guanidine hydrochloride or 8 M urea). The leader sequence and His-tag were cleaved by either cyanogen bromide (CNBr) under extreme acidic conditions or proteases (e.g. Factor Xa) under mild detergent conditions (e.g. Triton X-100). Finally, the CB2 fragments were purified by RP-HPLC. In the present manuscript, we reported that another CB2 fragment (CB2271-326), including part of the extracellular loop 3, TM7, helix 8 and part of the C-terminus based on the predicted 3D structural model of CB2 [16], can be produced according to the established methods generally described above. Some important modifications, as will be detailed below, were made to optimize the protein expression level, IB solubilization, fusion protein cleavage, product purification and refolding. The obtained pure CB2 fragment was characterized by circular dichroism and fluorescence spectroscopy. It has been known from the reported mutation and function studies that Ser285, Ser292 and Tyr299 in TM7 are important for CB2 ligand binding [17, 18]. The residue Tyr299 in TM7, as well as the conserved cysteins Cys313 and Cys320 in the C-terminal juxamembrane region were shown to be critical for coupling to adenylate cyclase [17]. The solution NMR structure of CB2271-326 fragment and the possible specific interaction of the fragment with G-protein will be examined in our future work. The current study provides useful information for structure/function studies of the fragment as well as the whole CB2 receptor.