Co-expression of silkworm allatostatin-C receptor BNGR-A1 with its cognate G protein subunits enhances the GPCR display on the budding baculovirus

Jianping Chen, Jian Xu, Masato Hino, Mami Yamashita, Kazuma Hirata, Anandrao Ashok Patil, Tsuneyuki Tatsuke, Hiroaki Mon, Yutaka Banno, Takahiro Kusakabe, Man Lee

研究成果: ジャーナルへの寄稿記事

2 引用 (Scopus)

抄録

G protein-coupled receptors (GPCRs) are seven transmembrane proteins, which play an essential role in transmitting various extracellular signals into cells. For functional and structural analysis of GPCRs, it is necessary to produce active GPCRs in high quantities with outstanding purity. Fortunately, earlier baculovirus expression vector system has been reported as a proven functional GPCR mass-production tool. Therefore, in this study, we selected Bombyx mori allatostatin-C neuropeptide receptor BNGR-A1 as a GPCR reporter protein, which has been already proved successful binding of ligand. We confirmed its expression profile in various silkworm tissues and cell lines and then verified its plasma membrane subcellular localization in cultured silkworm BmN4 cells. In addition, we constructed recombinant baculoviruses for BNGR-A1, its ligand allatostatin-C (BmAST-C) and related eight G proteins (Gs, G12, Gα4, Gq, Gβ2, Gβ3, Gβ5 and Gγ) and subsequently monitored the extracellular or intracellular expression of BNGR-A1 by co-infection with its ligand and cognate G proteins-expressing viruses. It is interesting to observe that different combinations of G proteins could result in changes or even undetectable of final yields of BNGR-A1, suggesting the essential roles of G proteins involved in the GPCR expression or stabilization. The present study demonstrated that co-infection of recombinant viruses expressing Gα4β3γ trimer enhanced the production of BNGR-A1 on BV fractions. To our knowledge, this is a fine strategy for identifying the specific G protein partner responsible for certain GPCR of interest. These studies would provide a novel idea for improving GPCR expression in the silkworm by baculovirus expression vector system.

元の言語英語
ページ(範囲)753-760
ページ数8
ジャーナルJournal of Asia-Pacific Entomology
19
発行部数3
DOI
出版物ステータス出版済み - 9 1 2016

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allatostatin
Baculoviridae
protein subunits
silkworms
G-proteins
receptors
mixed infection
viruses
transmembrane proteins
G-protein coupled receptors
Bombyx mori
purity
plasma membrane
cell lines
cells

All Science Journal Classification (ASJC) codes

  • Insect Science

これを引用

Co-expression of silkworm allatostatin-C receptor BNGR-A1 with its cognate G protein subunits enhances the GPCR display on the budding baculovirus. / Chen, Jianping; Xu, Jian; Hino, Masato; Yamashita, Mami; Hirata, Kazuma; Patil, Anandrao Ashok; Tatsuke, Tsuneyuki; Mon, Hiroaki; Banno, Yutaka; Kusakabe, Takahiro; Lee, Man.

:: Journal of Asia-Pacific Entomology, 巻 19, 番号 3, 01.09.2016, p. 753-760.

研究成果: ジャーナルへの寄稿記事

Chen, Jianping ; Xu, Jian ; Hino, Masato ; Yamashita, Mami ; Hirata, Kazuma ; Patil, Anandrao Ashok ; Tatsuke, Tsuneyuki ; Mon, Hiroaki ; Banno, Yutaka ; Kusakabe, Takahiro ; Lee, Man. / Co-expression of silkworm allatostatin-C receptor BNGR-A1 with its cognate G protein subunits enhances the GPCR display on the budding baculovirus. :: Journal of Asia-Pacific Entomology. 2016 ; 巻 19, 番号 3. pp. 753-760.
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abstract = "G protein-coupled receptors (GPCRs) are seven transmembrane proteins, which play an essential role in transmitting various extracellular signals into cells. For functional and structural analysis of GPCRs, it is necessary to produce active GPCRs in high quantities with outstanding purity. Fortunately, earlier baculovirus expression vector system has been reported as a proven functional GPCR mass-production tool. Therefore, in this study, we selected Bombyx mori allatostatin-C neuropeptide receptor BNGR-A1 as a GPCR reporter protein, which has been already proved successful binding of ligand. We confirmed its expression profile in various silkworm tissues and cell lines and then verified its plasma membrane subcellular localization in cultured silkworm BmN4 cells. In addition, we constructed recombinant baculoviruses for BNGR-A1, its ligand allatostatin-C (BmAST-C) and related eight G proteins (Gs, G12, Gα4, Gq, Gβ2, Gβ3, Gβ5 and Gγ) and subsequently monitored the extracellular or intracellular expression of BNGR-A1 by co-infection with its ligand and cognate G proteins-expressing viruses. It is interesting to observe that different combinations of G proteins could result in changes or even undetectable of final yields of BNGR-A1, suggesting the essential roles of G proteins involved in the GPCR expression or stabilization. The present study demonstrated that co-infection of recombinant viruses expressing Gα4β3γ trimer enhanced the production of BNGR-A1 on BV fractions. To our knowledge, this is a fine strategy for identifying the specific G protein partner responsible for certain GPCR of interest. These studies would provide a novel idea for improving GPCR expression in the silkworm by baculovirus expression vector system.",
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T1 - Co-expression of silkworm allatostatin-C receptor BNGR-A1 with its cognate G protein subunits enhances the GPCR display on the budding baculovirus

AU - Chen, Jianping

AU - Xu, Jian

AU - Hino, Masato

AU - Yamashita, Mami

AU - Hirata, Kazuma

AU - Patil, Anandrao Ashok

AU - Tatsuke, Tsuneyuki

AU - Mon, Hiroaki

AU - Banno, Yutaka

AU - Kusakabe, Takahiro

AU - Lee, Man

PY - 2016/9/1

Y1 - 2016/9/1

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AB - G protein-coupled receptors (GPCRs) are seven transmembrane proteins, which play an essential role in transmitting various extracellular signals into cells. For functional and structural analysis of GPCRs, it is necessary to produce active GPCRs in high quantities with outstanding purity. Fortunately, earlier baculovirus expression vector system has been reported as a proven functional GPCR mass-production tool. Therefore, in this study, we selected Bombyx mori allatostatin-C neuropeptide receptor BNGR-A1 as a GPCR reporter protein, which has been already proved successful binding of ligand. We confirmed its expression profile in various silkworm tissues and cell lines and then verified its plasma membrane subcellular localization in cultured silkworm BmN4 cells. In addition, we constructed recombinant baculoviruses for BNGR-A1, its ligand allatostatin-C (BmAST-C) and related eight G proteins (Gs, G12, Gα4, Gq, Gβ2, Gβ3, Gβ5 and Gγ) and subsequently monitored the extracellular or intracellular expression of BNGR-A1 by co-infection with its ligand and cognate G proteins-expressing viruses. It is interesting to observe that different combinations of G proteins could result in changes or even undetectable of final yields of BNGR-A1, suggesting the essential roles of G proteins involved in the GPCR expression or stabilization. The present study demonstrated that co-infection of recombinant viruses expressing Gα4β3γ trimer enhanced the production of BNGR-A1 on BV fractions. To our knowledge, this is a fine strategy for identifying the specific G protein partner responsible for certain GPCR of interest. These studies would provide a novel idea for improving GPCR expression in the silkworm by baculovirus expression vector system.

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