Efficient protein production using a Bombyx mori nuclear polyhedrosis virus lacking the cysteine proteinase gene.

Suzuki, Takeo; Kanaya, Toshimichi; Okazaki, Hironobu; Ogawa, Katsuaki; Usami, Akihiro; Watanabe, Hitoshi; Kadono-Okuda, Keiko; Yamakawa, Minoru; Sato, Hideki; Mori, Hajime; Takahashi, Saori; Oda, Kōhei

doi:10.1099/0022-1317-78-12-3073

Cited by 99 publications

(44 citation statements)

References 26 publications

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“…Different types of protease from those in Sf-9 cells may exist in Tn-5B1-4 cells. The addition of protease (carboxyl and cystein protease) inhibitors to a culture and the use of a protease-deficient recombinant baculovirus should contribute to more efficient production of the GFP uv -␤3GnT2 fusion protein [24][25][26][27].…”

Section: Discussionmentioning

confidence: 99%

Efficient production of human β-1,3-N-acetylglucosaminyltransferase-2 fused with green fluorescence protein in insect cell

Kato

Murata

Usui

et al. 2004

Biochemical Engineering Journal

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Human ␤-1,3-N-acetylglucosaminyltransferase-2 (␤3GnT2) was produced in a baculovirus expression system as a secreted fusion protein with a green fluorescence protein variant, GFP uv , flanked by the (His) 6 sequence and an enterokinase cleavage site. The expression of the ␤3GnT2-GFP uv fusion gene was rapidly detected using a fluorescence microscope without employing complicated assay methods. When Tn-5B1-4 cells were infected with a recombinant AcMNPV-␤3GnT2-GFP uv virus at MOI 10, intracellular and extracellular ␤3GnT activities increased to 0.26 and 0.68 mU/ml, respectively, until 3 days post-infection (d.p.i.), and decreased markedly at 3 d.p.i. In contrast to Tn-5B1-4 cell culture medium, the extracellular ␤3GnT activity in Sf-9 cell culture medium increased to 0.86 mU/ml at 4 d.p.i. The fusion protein obtained from Tn-5B1-4 and Sf-9 cultures was confirmed based on the GFP uv of the fusion protein. The fusion protein was purified using a Ni 2+ affinity column, and was concentrated by approximately 900-fold. The observed ␤3GnT activity and the specific ␤3GnT activity of the purified fusion protein were 77.6 mU/ml and 4.6 U/mg protein, respectively. When the purified fusion protein was treated with glycopeptidase F, its molecular weight decreased by 7-8 kDa, indicating that ␤3GnT2 is glycosylated.

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Section: Discussionmentioning

confidence: 99%

Efficient production of human β-1,3-N-acetylglucosaminyltransferase-2 fused with green fluorescence protein in insect cell

Kato

Murata

Usui

et al. 2004

Biochemical Engineering Journal

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“…BmNPV codes cysteine protease and the activity is detected in the BmNPV-infected silkworm larval hemolymph [18,19]. However, the cysteine protease may not be involved in the case of canine interferon-γ, because the generation of CaIFN-γ/15(-), CaIFN-γ/16(-), and CaIFN-γ/17(-) is inhibited at pH 4 and stimulated under alkaline condition.…”

Section: Degradation Of C-terminal Endmentioning

confidence: 99%

Manufacturing Pharmaceutical-Grade Interferons Using Silkworm- Baculovirus System

Tanaka¹

2012

J Biotechnol Biomaterial

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To develop feline and canine interferons as medicine for companion animal, silkworm-baculovirus expression system was established as a manufacturing technology. Thus produced interferons were successfully cleared for a sale in commercial market. For expression of feline interferon in the silkworm, recombinant Bombyx mori nucleopolyhedrovirus (BmNPV) was constructed carrying feline interferon cDNA. When the recombinant BmNPV was injected to silkworm larvae, as much as 1.2 × 10 8 unit/mL of feline interferon was produced in their hemolymph. This productivity is 100-fold higher than those of E. coli, yeast and mammalian cell expression system. Similarly, canine interferon-γ was produced in the largest amount in silkworm. Two or more forms of interferons were produced due to various processing in silkworm. For feline interferon, two forms of protein molecule resulting from two different cleavage sites in signal peptide sequence was solved by reconstitution of a single amino acid in signal sequence. In the case of canine interferon-γ, several variations were produced due to glycosylation pattern and C-terminal degradation, but removal of glycosylation sites and elimination of C-terminal 16 amino acids sequence from native sequence lead to uniformity of the expressed protein. Drug formulated two recombinant animal interferons were approved as a treatment of viral diseases and atopic dermatitis by regulatory authorities. Especially, feline interferon is now sold in 20 countries including Japan and Europe.

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“…The PCR fragments of merF or merThis tag were inserted into the XhoI-XbaI site in the transfer vector pM01 (Katakura Industries Co., Ltd., Saitama, Japan) and sequenced. The transfer vector and linearized genomic DNA from the ABv baculovirus (Bombyx mori nucleopolyhedrovirus; CPd strain; Katakura Industries Co., Ltd., Saitama, Japan) 22) were co-transfected into B. mori-cultured cells (BmN). 23) After propagating the recombinant baculovirus containing the merF-or merT-his 6 -tagged gene in BmN cells, the virus was used to infect silkworm pupae.…”

Section: Computer Analyses Of Protein Sequencesmentioning

confidence: 99%

Role of MerC, MerE, MerF, MerT, and/or MerP in Resistance to Mercurials and the Transport of Mercurials in <i>Escherichia coli</i>

Sone

Nakamura

Pan-Hou

et al. 2013

Biological & Pharmaceutical Bulletin

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The characteristics of bacteria take up mercury into cells via membrane potential-dependent sequencedivergent members of the mercuric ion (Mer) superfamily, i.e., a periplasmic mercuric ion scavenging protein (MerP) and one or more inner membrane-spanning proteins (MerC, MerE, MerF, and MerT), which transport mercuric ions into the cytoplasm, have been applied in engineering of bioreactor used for mercurial bioremediation. We engineered bacteria to express MerC, MerE, MerF, or MerT with or without MerP to clarify their individual role and potential in transport of mercurial. By immunoblot analysis using specific polyclonal antibody, the proteins encoded by merC, merE, merF, merT or merP, were certainly expressed and identified in the membrane fraction. Bacteria expressing MerC, MerE, MerF or MerT in the absence of MerP transported significantly more C 6 H 5 Hg(I) and Hg(II) across bacterial membrane than their isogenic strain. In vivo expression of MerP in the presence of all the transporters did not cause apparent difference to the C 6 H 5 Hg(I) transport, but gives an apparently higher Hg(II) transport than that did by MerE, MerF or MerT but not by MerC. Among the four transporters studied, MerC showed more potential to transport Hg(II) across bacterial membrane than MerE, MerF and MerT. Together these findings, we demonstrated for the first time that in addition to MerE and MerT, MerF and MerC are broad-spectrum mercury transporters that mediate both Hg(II) and phenylmercury transport into cells. Our results suggested that MerC is the most efficient tool for designing mercurial bioremediation systems, because MerC is sufficient for mercurial transport into cells.

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Efficient protein production using a Bombyx mori nuclear polyhedrosis virus lacking the cysteine proteinase gene.

Cited by 99 publications

References 26 publications

Efficient production of human β-1,3-N-acetylglucosaminyltransferase-2 fused with green fluorescence protein in insect cell

Efficient production of human β-1,3-N-acetylglucosaminyltransferase-2 fused with green fluorescence protein in insect cell

Manufacturing Pharmaceutical-Grade Interferons Using Silkworm- Baculovirus System

Role of MerC, MerE, MerF, MerT, and/or MerP in Resistance to Mercurials and the Transport of Mercurials in <i>Escherichia coli</i>

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