Efficient silkworm expression of human GPCR (nociceptin receptor) by a Bombyx mori bacmid DNA system

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Abstract

Guanine nucleotide-binding protein (G protein) coupled receptors (GPCRs) are frequently expressed by a baculovirus expression vector system (BEVS). We recently established a novel BEVS using the bacmid system of Bombyx mori nucleopolyhedrovirus (BmNPV), which is directly applicable for protein expression in silkworms. Here, we report the first example of GPCR expression in silkworms by the simple injection of BmNPV bacmid DNA. Human nociceptin receptor, an inhibitory GPCR, and its fusion protein with inhibitory G protein alpha subunit (Giα) were both successfully expressed in the fat bodies of silkworm larvae as well as in the BmNPV viral fraction. Its yield was much higher than that from Sf9 cells. The microsomal fractions including the nociceptin receptor fusion, which are easily prepared by only centrifugation steps, exhibited [35S]GTPγS-binding activity upon specific stimulation by nociceptin. Therefore, this rapid method is easy-to-use and has a high expression level, and thus will be an important tool for human GPCR production.

Introduction

Guanine nucleotide-binding protein (G protein) coupled receptors (GPCR) are cell surface membrane proteins that regulate a broad range of physiological functions related to numerous diseases [1]. Thus, many widely used drugs directly target GPCR-mediated signaling. GPCRs have seven transmembrane regions, and associate with trimeric G protein, composed of alpha (Gα), beta (Gβ), and gamma (Gγ) subunits, at the cytoplasmic region [2]. The binding of specific ligands (peptides, lipids, steroids, etc.) to the extracellular region of GPCRs induces the enhancement of the GTPase activity of trimeric G protein, to exchange Gα-bound GTP to GDP. This hydrolysis leads to the dissociation of trimeric G protein subunits from GPCRs, and this triggers the following G protein-mediated signal transduction [3].

To date, several hundred GPCR family genes have been identified in the human genome [4]. However, the physiological ligands of many GPCRs remain unknown, and thus they are designated as “orphan receptors”[5]. Many researchers are searching for the physiological GPCR ligands, to understand their functions and to develop agonists or antagonists as effective, new drugs. In order to perform ligand screening and biochemical studies, large amounts of GPCR proteins are required. However, the expression of GPCRs is still difficult, thus poses a major obstacle for GPCR studies.

Escherichia coli (E. coli) expression systems are frequently used for the large-scale expression of recombinant proteins. However, E.coli systems have critical disadvantages for the expression of eukaryotic proteins, such as the lack of intramolecular disulfide bond formation and post-translational modifications. In the case of GPCRs, a small number of groups reported the successful expression of recombinant GPCRs using E. coli. [6], [7], [8]. Many researchers prefer eukaryotic expression systems to produce GPCRs. The baculovirus expression vector system (BEVS) is one of the attractive tools for the large-scale expression of recombinant GPCRs. BEVS has many advantages for the expression of eukaryotic proteins, including (1) high level expression by strong promoters (Polyhedrin and P10), and (2) post-translational modifications similar to those generated by mammalian cell expression. BEVS was employed in the successful crystallization of human adrenergic receptor protein for structure determination [9], [10], [11], [12]. However, the commercially available BEVS system, which utilizes Autographa californica nucleopolyhedrovirus (AcNPV) and insect cell lines (High five™, Sf9 and Sf21), requires large-scale cultivation and virus handling techniques.

Recently, we established a novel and effective BEVS using Bombyx mori nucleopolyhedrovirus (BmNPV) bacmid DNA, which is directly applicable to silkworm expression [13]. The bacmid DNA is the baculovirus shuttle vector system based on the BmNPV genomic DNA. This bacmid DNA can replicate in E. coli, and the direct injection of its mixture with a cationic lipid reagent induces the expression of recombinant proteins in silkworm larvae. We successfully demonstrated the abundant expression of green fluorescent protein (GFP) in silkworm larvae, using this BEVS system [13]. Moreover, we improved the expression level by deleting the chitinase and cysteine protease genes from the BmNPV bacmid DNA [14], [15].

Nociceptin, an exogenous opioid peptide, plays an important role in pain control [16]. The nociceptin receptor (NR) is a typical seven transmembrane GPCR that binds nociceptin to regulate the neurological response. The cytoplasmic region of NR associates with an inhibitory trimeric G protein including an inhibitory Gα subunit (Giα). Nociceptin binding to NR induces the activation of GTP hydrolysis by Giα to mediate the inhibitory signal, and thus NR is classified as an “inhibitory” GPCR. To monitor the functions of the activating GPCRs that associate with the activating Gα subunits, Gs and Gq, the intracellular Ca2+ or cAMP levels, which can be increased by the ligand-induced stimulation, are frequently determined. However, these assays are not directly applicable to the inhibitory GPCRs, because these GPCRs block activation signals. In order to estimate the inhibitory signals, the measurement of radiolabeled GTP agonist binding to Giα is employed. To ensure the efficient and stable association between Gα and GPCR, chimeras with Gα fused to the C-terminus of the GPCR are utilized [17], [18], [19], [20]. Our previous reports demonstrated that the functional Giα-fused NRs could be expressed in Sf9 cells [19], [20] .

We now report the first example of the production of human inhibitory GPCR protein, by the combination of the BmNPV bacmid technique and silkworm expression. The recombinant BmNPV bacmid DNAs encoding human NR and its variants were prepared in bacteria. They were mixed with a lipid reagent and were simply injected in silkworm larvae. Seven days after the injection, NRs as well as the BmNPV virus were expressed on the fat bodies of the silkworms. Small amounts of microsomal fractions of Giα-fused NR, isolated from the fat bodies, showed specific [35S]GTPγS-binding activity. Based on these results, we discuss the advantages of this BmNPV bacmid–silkworm expression system, as compared with other systems, for future GPCR production.

Section snippets

Materials and methods

Insect and antibodies. Silkworms (B.mori, Kinshu × Showa race) were used as an expression host. Newly ecdysed fifth instar larvae were purchased from Nosan Corporation (Yokohama, Japan) and were cultivated with an artificial diet (Nosan Corporation) at 25 °C. For Western blotting, an anti-penta-His antibody (34660; QIAGEN, Hilden, Germany) was used as the primary antibody, and horseradish peroxidase (HRP)-conjugated sheep anti-mouse IgG (NA931; GE healthcare, Little Chalfont, UK) was used as the

Results and discussion

Nociceptin receptor (NR) is an “inhibitory” GPCR. Inhibitory trimeric G protein, which includes the inhibitory Gα subunit (Giα), associates with the cytoplasmic region of NR. Nociceptin binding to NR induces the stimulation of the GTP hydrolytic activity of Giα, resulting in the mediation of inhibitory signals, which are normally more difficult to detect than activating signals (e.g. the determination of the intracellular Ca2+ or cAMP levels). In order to easily monitor the activation of

Acknowledgments

We thank M. Sasaki, M. Ohtsu, and D. Kohda (Medical Institute of Bioregulation) and stuff of the Research Support Center, Graduate School of Medical Sciences, Kyushu University. K.M. was supported in part by the Ministry of Education, Culture, Sports, Science and Technology and the Ministry of Health, and the Japan Bio-oriented Technology Research Advancement Institute (BRAIN).

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