Expression, purification, and characterization of highly active endo-α-N-acetylgalactosaminidases expressed by silkworm-baculovirus expression system

Morio, Akihiro; Xu, Jian; Masuda, Akihiro; Kinoshita, Yurie; Hino, Masato; Morokuma, Daisuke; Goda, Hatsumi M.; Okino, Nozomu; Ito, Makoto; Mon, Hiroaki; Fujita, Ryosuke; Kusakabe, Takahiro; Lee, Jae Man

doi:10.1016/j.aspen.2019.01.009

Cited by 5 publications

(2 citation statements)

References 21 publications

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“…In this chromatography, BmPvs25 can be eluted remarkably at 100 mM elution concentration by imidazole, but silkworm-derived proteins were also eluted at the same condition. Since it is known from previous studies that silkworm-derived proteins (about 70 kDa) bind with Ni-immobilized metal affinity resin [38], we further purified BmPvs25 with StrepTactin affinity column. The fractions of Ni-affinity chromatography containing BmPvs25 were collected (lanes 4 in Figure 2) and applied to StrepTactin column.…”

Section: Resultsmentioning

confidence: 99%

Highly efficient protein expression of Plasmodium vivax surface antigen, Pvs25 by silkworm, Bombyx mori, and its biochemical analysis

Miyata

Minamihata

Kurihara

et al. 2022

Preprint

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Plasmodium vivax ookinete surface protein, Pvs25 is a transmission-blocking vaccine (TBV) candidate for malaria. Pvs25 has four EGF-like domains containing 22 cysteine residues forming 11 intramolecular disulfide bonds and this structural feature makes recombinant expression of Pvs25 difficult. In this study, we report the high expression of recombinant Pvs25 as a soluble form in silkworm, Bombyx mori. The Pvs25 protein was purified from hemolymphs of larvae and pupae by affinity chromatography. In the Pvs25 expressed by silkworm, no isoform with inappropriate disulfide bonds was found, requiring no further purification step which is necessary in case of Pichia pastoris based expressions systems. The Pvs25 from silkworm were confirmed to be the molecularly uniform by sodium dodecyl sulfate gel electrophoresis analysis and size exclusion chromatography analysis. To examine the immunogenicity, the Pvs25 from B. mori, was administered to BALB/c mice by the subcutaneous (s.c.) route with the oil adjuvant. The Pvs25 produced by silkworm induced potent and robust immune response, and the induced antisera correctly recognized P. vivax ookinetes in vitro, demonstrating the potency of Pvs25 from silkworm as a TBV candidate for malaria. This is the first study that to construct a mass production system for malaria TBV antigens by the silkworm to the best of our knowledge.

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Section: Resultsmentioning

confidence: 99%

Highly efficient protein expression of Plasmodium vivax surface antigen, Pvs25 by silkworm, Bombyx mori, and its biochemical analysis

Miyata

Minamihata

Kurihara

et al. 2022

Preprint

Self Cite

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“…Enterococcus faecalis strains possess genes encoding endo- β - N -acetylglucosaminidase and endo- α - N -acetylgalactosaminidase (endo-α-GalNAc-ase) that release N -glycans. However, this pathobiont appears not to be able to utilize mucin O -linked glycans ( Roberts et al, 2000 ; Morio et al, 2019 ). These microorganisms are not usually dominant in the gut microbiota, probably due to the barrier effect mounted by this community.…”

Section: Utilization Of Mucin O -Glycans By Gut MImentioning

confidence: 99%

Molecular Insights Into O-Linked Glycan Utilization by Gut Microbes

2020

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O-linked glycosylation is a post-translational modification found mainly in eukaryotic cells, which covalently attaches oligosaccharides to secreted proteins in certain threonine or serine residues. Most of O-glycans have N-acetylgalactosamine (GalNAc) as a common core. Several glycoproteins, such as mucins (MUCs), immunoglobulins, and caseins are examples of O-glycosylated structures. These glycans are further elongated with other monosaccharides and sulfate groups. Some of them could be found in dairy foods, while others are produced endogenously, in both cases interacting with the gut microbiota. Interestingly, certain gut microbes can access, release, and consume O-linked glycans as a carbon source. Among these, Akkermansia muciniphila, Bifidobacterium bifidum, and Bacteroides thetaiotaomicron are prominent O-linked glycan utilizers. Their consumption strategies include specialized α-fucosidases and α-sialidases, in addition to endo-α-N-acetylgalactosaminidases that release galacto-N-biose (GNB) from peptides backbones. O-linked glycan utilization by certain gut microbes represents an important niche that allows them to predominate and modulate host responses such as inflammation. Here, we focus on the distinct molecular mechanisms of consumption of O-linked GalNAc glycans by prominent gut microbes, especially from mucin and casein glycomacropeptide (GMP), highlighting the potential of these structures as emerging prebiotics.

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Expression and Purification of Vaccinia Virus DNA Topoisomerase IB Produced in the Silkworm–Baculovirus Expression System

Lee

Tatsuke

et al. 2019

Mol Biotechnol

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Expression, purification, and characterization of highly active endo-α-N-acetylgalactosaminidases expressed by silkworm-baculovirus expression system

Cited by 5 publications

References 21 publications

Highly efficient protein expression of Plasmodium vivax surface antigen, Pvs25 by silkworm, Bombyx mori, and its biochemical analysis

Highly efficient protein expression of Plasmodium vivax surface antigen, Pvs25 by silkworm, Bombyx mori, and its biochemical analysis

Molecular Insights Into O-Linked Glycan Utilization by Gut Microbes

Expression and Purification of Vaccinia Virus DNA Topoisomerase IB Produced in the Silkworm–Baculovirus Expression System

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