Production of Canine IFN-γ in Silkworm by Recombinant Baculovirus and Characterization of the Product

Okano, Fumiyoshi; Satoh, Masahiro; Ido, Takayoshi; Okamoto, Naoyo; Yamada, Katsushige

doi:10.1089/10799900050198462

Cited by 16 publications

(12 citation statements)

References 23 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…Most reported insect/baculovirus systems are based on Bombyx mori nucleopolyhedrovirus (BmNPV) infecting silkworms (Bombyx mori); silkworms are large and easily reared, and BmNPV has a restricted pathogenicity. In Japan, both small and large scale commercial protein productions are carried out in B. mori larvae, (http://www.katakura.co.jp/ superworm; http://www.toray.jp/chemical/english [10]). …”

Section: Introductionmentioning

confidence: 99%

Expression of two heterologous proteins depends on the mode of expression: comparison of in vivo and in vitro methods

Gatehouse

Markwick

Poulton

et al. 2008

Bioprocess Biosyst Eng

View full text Add to dashboard Cite

The yield of two proteins, avidin and green fluorescent protein (GFP), expressed from a modified Autographa californica nucleopolyhedrovirus (AcMNPV), was compared in Sf9 cell culture monolayer, Sf21 cell suspension culture and intact Spodoptera litura larvae. GFP expressed from the p10 promoter yielded up to 1.5% of total soluble protein in larvae, 20-fold higher than that in monolayer suspension culture. Avidin, expressed from the polh promoter, yielded up to 2.3% of total soluble protein in larvae, 10-fold higher than that in suspension culture and 40-fold higher than that in monolayers. Avidin expression did not affect amounts of GFP in dual-expressing baculovirus compared with those detected from a GFP-only expressing AcMNPV. A biotin-binding assay showed that all avidin expressed in larvae was fully active. Glycosylation patterns of chicken-avidin and Spodoptera-avidin were very similar, though the latter showed a proportion of partially glycosylated material.

show abstract

Section: Introductionmentioning

confidence: 99%

Expression of two heterologous proteins depends on the mode of expression: comparison of in vivo and in vitro methods

Gatehouse

Markwick

Poulton

et al. 2008

Bioprocess Biosyst Eng

View full text Add to dashboard Cite

show abstract

“…Silkworm expression system showed the highest productivity as well as feline interferon. The expressed canine interferon-γ in silkworm hemolymph showed four main bands in western blotting with anti-interferon-γ antiserum ( Figure 3C), lane 1), because it formed complex protein [17]. There are two N-glycosylation sites in the canine interferon-γ which means that the presence of different bands might be due to the difference of N-glycosylation.…”

Section: Expression Of Canine Interferon-γ In Silkworm Larvaementioning

confidence: 97%

“…From the N-terminal amino acid sequence, signal peptide was cleaved correctly at single site and the purified CaIFN-γ/16(-) had the characteristic bioactivity of canine interferon-γ represented by anti-virus activity, anti-tumor activity against canine neoplastic cells, and facilitation activity of the expression of MHC class II antigen [17]. The specific activity was 9.0 × 10 7 unit/mg, which is comparable to that of non truncated canine interferon-γ [17].…”

Section: Degradation Of C-terminal Endmentioning

confidence: 99%

Manufacturing Pharmaceutical-Grade Interferons Using Silkworm- Baculovirus System

Tanaka¹

2012

J Biotechnol Biomaterial

View full text Add to dashboard Cite

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.

show abstract

“…The B. mori nucleopolyhedrosis virus (BmNPV) expression system can take advantage of the inexpensive, convenient, and high-level production of heterologous proteins in silkworm larvae. This expression system has been successfully applied to produce a large amount of foreign proteins such as human vascular endothelial growth factor [12] and different interferon isoforms from bovine or canine species [13][14][15]. Therefore, the baculovirus expression system might be considered one of the most effective expression tools in silkworm larvae [15,16]; however, its use to infect chrysalides of this insect is less known.…”

Section: Introductionmentioning

confidence: 99%

Purification and Kinetic Properties of Human Recombinant Dihydrofolate Reductase Produced in Bombyx mori Chrysalides

Chazarra

Aznar-Cervantes

Sánchez‐del‐Campo

et al. 2010

Appl Biochem Biotechnol

View full text Add to dashboard Cite

Recent reports describe the inhibition of human dihydrofolate reductase (hDHFR) by natural tea polyphenols. This finding could explain the epidemiologic data on their prophylactic effects for certain forms of cancer, and it raises the possibility that natural and synthetic polyphenols could be used in cancer chemotherapy. In order to obtain larger quantities of hDHFR to support structural studies, we established and validated a baculovirus system for the expression of this protein in Bombyx mori chrysalides (pupae of the silkworm enclosed in a cocoon). To isolate the expressed protein, whole infected pupae were homogenized, and the expressed protein was purified by affinity chromatography. Here, we demonstrate the efficient expression of recombinant hDHFR in this model and report that this newly expressed protein has high enzymatic activity and kinetic properties similar to those previously reported for recombinant hDHFR expressed in Escherichia coli. The purified protein showed dissociation constants for the binding of natural polyphenols similar to that expressed in E. coli, which ensures its usage as a new tool for further structural studies. Although the hDHFR yield per individual was found to be lower in the chrysalides than in the larvae of B. mori, the former system was optimized as a model for the scaled-up production of recombinant proteins. Expression of proteins in chrysalides (instead of larvae) could offer important advantages from both economic and biosecurity aspects.

show abstract

Production of Canine IFN-γ in Silkworm by Recombinant Baculovirus and Characterization of the Product

Cited by 16 publications

References 23 publications

Expression of two heterologous proteins depends on the mode of expression: comparison of in vivo and in vitro methods

Expression of two heterologous proteins depends on the mode of expression: comparison of in vivo and in vitro methods

Manufacturing Pharmaceutical-Grade Interferons Using Silkworm- Baculovirus System

Purification and Kinetic Properties of Human Recombinant Dihydrofolate Reductase Produced in Bombyx mori Chrysalides

Contact Info

Product

Resources

About