Active Human and Murine Tumor Necrosis Factor α Cytokines Produced from Silkworm Baculovirus Expression System

Ebihara, Takeru; Xu, Jian; Tonooka, Yoshino; Nagasato, Takumi; Kakino, Kohei; Masuda, Akihiro; Minamihata, Kosuke; Kamiya, Noriho; Nakatake, Hirokazu; Chieda, Yuuka; Mon, Hiroaki; Fujii, Tsuguru; Kusakabe, Takahiro; Lee, Jae Man

doi:10.3390/insects12060517

Cited by 3 publications

(2 citation statements)

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“…Each resulting plasmid was then transformed into E. coli BmDH10Bac to construct a recombinant B. mori nucleopolyhedrovirus (BmNPV) containing the VP6 gene [ 26 ]. VP6 was then attached with the signal peptide of 30k6G protein that encodes a 30 kDa lipoprotein in silkworms [ 27 ], to obtain 30k-T-VP6 (Fig. 1 ).…”

Section: Methodsmentioning

confidence: 99%

Expression and Purification of Porcine Rotavirus Structural Proteins in Silkworm Larvae as a Vaccine Candidate

et al. 2022

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In this study, silkworm larvae were used for expression of porcine rotavirus A (KS14 strain) inner capsid protein, VP6, and outer capsid protein, VP7. Initially, VP6 was fused with Strep-tag II and FLAG-tag (T-VP6), and T-VP6 was fused further with the signal peptide of Bombyx mori 30k6G protein (30k-T-VP6). T-VP6 and 30 k-T-VP6 were then expressed in the fat body and hemolymph of silkworm larvae, respectively, with respective amounts of 330 μg and 50 μg per larva of purified protein. Unlike T-VP6, 30k-T-VP6 was N -glycosylated due to attached signal peptide. Also, VP7 was fused with PA-tag (VP7-PA). Additionally, VP7 was fused with Strep-tag II, FLAG-tag, and the signal peptide of Bombyx mori 30k6G protein (30k-T-ΔVP7). Both VP7-PA and 30k-T-ΔVP7 were expressed in the hemolymph of silkworm larvae, with respective amounts of 26 μg and 49 μg per larva of purified protein, respectively. The results from our study demonstrated that T-VP6 formed nanoparticles of greater diameter compared with the ones formed by 30k-T-VP6. Also, higher amount of VP6 expressed in silkworm larvae reveal that VP6 holds the potential for its use in vaccine development against porcine rotavirus with silkworm larvae as a promising host for the production of such multi-subunit vaccines.

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Section: Methodsmentioning

confidence: 99%

Expression and Purification of Porcine Rotavirus Structural Proteins in Silkworm Larvae as a Vaccine Candidate

et al. 2022

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“…As a result of increasing demand for sTNF-α protein, recombinant human TNF-α protein with appropriate bioactivities was produced in several heterologous protein expression systems such as Escherichia coli [ 13 , 14 ], Lactococcus lactis [ 15 ], Streptomyces lividans [ 16 ], a baculovirus expression system [ 17 , 18 ], or mouse embryonic fibroblasts [ 19 ]. Continuous efforts have been made to maximize TNF-α productivity from an E. coli expression system, including gene codon optimization, choice of the strongest expression promoters and optimization of culture and induction conditions to obtain more soluble recombinant protein over inclusion bodies [ 13 , 14 , 20 , 21 , 22 ].…”

Section: Introductionmentioning

confidence: 99%

A De Novo Optimized Cell-Free System for the Expression of Soluble and Active Human Tumor Necrosis Factor-Alpha

El-Baky

El‐Fakharany

Sabry

et al. 2022

Biology

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Cell-free (in vitro) expression is a robust alternative platform to the cell-based (in vivo) system for recombinant protein production. Tumor necrosis factor-alpha (TNF-α) is an effective pro-inflammatory cytokine with pleiotropic effects. The aim of the current study was de novo optimized expression of soluble and active human TNF-α by an in vitro method in an E. coli-based cell-free protein synthesis (CFPS) system and its biological activity evaluation. The codon-optimized synthetic human TNF-α gene was constructed by a two-step PCR, cloned into pET101/D-TOPO vector and then expressed by the E. coli CFPS system. Cell-free expression of the soluble protein was optimized using a response surface methodology (RSM). The anticancer activity of purified human TNF-α was assessed against three human cancer cell lines: Caco-2, HepG-2 and MCF-7. Data from RSM revealed that the lowest value (7.2 µg/mL) of cell-free production of recombinant human TNF-α (rhTNF-α) was obtained at a certain incubation time (6 h) and incubation temperature (20 °C), while the highest value (350 µg/mL) was recorded at 4 h and 35 °C. This rhTNF-α showed a significant anticancer potency. Our findings suggest a cell-free expression system as an alternative platform for producing soluble and functionally active recombinant TNF-α for further research and clinical trials.

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Production of antiviral vaccine antigens using a silkworm-baculovirus expression system

Kusakabe

2023

Journal of Pharmacological Sciences

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Active Human and Murine Tumor Necrosis Factor α Cytokines Produced from Silkworm Baculovirus Expression System

Cited by 3 publications

References 45 publications

Expression and Purification of Porcine Rotavirus Structural Proteins in Silkworm Larvae as a Vaccine Candidate

Expression and Purification of Porcine Rotavirus Structural Proteins in Silkworm Larvae as a Vaccine Candidate

A De Novo Optimized Cell-Free System for the Expression of Soluble and Active Human Tumor Necrosis Factor-Alpha

Production of antiviral vaccine antigens using a silkworm-baculovirus expression system

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