Secretory production of single-chain antibody (scFv) in Brevibacillus choshinensis using novel fusion partner

Tokunaga, Masao; Mizukami, Masaaki; Yamasaki, Koji; Tokunaga, Hiroko; Onishi, Hiromasa; Hanagata, Hiroshi; Ishibashi, Matsujiro; Miyauchi, Akira; Tsumoto, Kouhei; Arakawa, Tsutomu

doi:10.1007/s00253-013-4695-2

Cited by 19 publications

(13 citation statements)

References 39 publications

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“…To the best of our knowledge, there are few medium used for Brevibacillus choshinensis fermentation, which mainly includes 2SY [22,23], TM [22,23,31,32], and TM-derived medium [36]. When expressed using 2SY medium, the pullulanase activity was 24.3 U/mL, which was only 49.2 % of that obtained with TM medium.…”

Section: Optimization Of the Culture Medium Employing Single-factor Rmentioning

confidence: 88%

Efficient extracellular expression of Bacillus deramificans pullulanase in Brevibacillus choshinensis

Zou

Duan

2016

Journal of Industrial Microbiology and Biotechnology

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In this study, the pullulanase gene from Bacillus deramificans was efficiently expressed in Brevibacillus choshinensis. The optimal medium for protein expression was determined through a combination of single-factor experiments and response surface methodology. The initial pH of the medium and the culture temperature were optimized. The pullulanase yield increased 10.8-fold through medium and condition optimization at the shake-flask level. From the results of these experiments, the dissolved oxygen level was optimized in a 3-L fermentor. Under these optimized conditions, the pullulanase activity and the specific pullulanase productivity reached 1005.8 U/mL and 110.5 × 10(3) U/g dry cell weight, respectively, with negligible intracellular expression. The Brevibacillus choshinensis expression system has proven to be valuable for the extracellular production of pullulanase.

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Section: Optimization Of the Culture Medium Employing Single-factor Rmentioning

confidence: 88%

Efficient extracellular expression of Bacillus deramificans pullulanase in Brevibacillus choshinensis

Zou

Duan

2016

Journal of Industrial Microbiology and Biotechnology

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“…Although various partner proteins have been developed, "halophilic proteins" possess unique characteristics suitable as a fusion partner, including their high solubility, difficulty in salting out and ability to reversibly unfold and refold under denaturation. In the Brevibacillus-based expression production of scFv molecules, halophilic β-lactamase (BLA) was tested as an expression partner [22]. Anti-hen egg white lysozyme-scFv antibody (scFvHEL) was expressed and secreted as a BLA fusion protein (BLA-scFv-His-tag).…”

Section: Efficient Production Of Scfv Fragment [17]mentioning

confidence: 99%

Efficient Expression of Antibody Fragments with the Brevibacillus Expression System

Hanagata¹,

Mizukami²,

Miyauchi³

2014

Antibodies

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Antibodies, owing to their capability to bind specifically to a target molecule, have been and will continue to be applied in various areas, including research, diagnosis and therapy. In particular, antibody fragments, which are size-reduced antibodies comprising functional variable domains, are suited for production in bacteria. They also are useful in applications requiring intracellular delivery and for further engineering toward molecules possessing multiple custom functions. An expression system based on Brevibacillus is characterized by high efficiency and simple genetic recombination for secretory production. The Brevibacillus expression system has been successfully utilized for the efficient production of antibody fragments, e.g., scFvs (single-chain antibody fragments) comprising heavy-chain and light-chain variable domains, linked by a spacer sequence. Expression in fusion with a Halobacterium-derived secretory protein was shown to confer enhanced productivity. In the case of Fabs, productivity as high as 100 mg/L was accomplished in a simple system, i.e., shake flask cultures. The Brevibacillus expression system offers several advantages, shared by other bacterial systems, such as E. coli, in particular, for the ease in genetic engineering and culture production.

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“…In addition, this expression system is very powerful for producing proteins with native structures, even when they contain disulfide bonds (23). pNCMO2 is a B. choshinensis-E. coli shuttle vector (24). The pNCMO2 vector includes the P2 promoter, which drives the transcription of cell wall protein but has no role in E. coli (24).…”

Section: Introductionmentioning

confidence: 99%

Extracellular production of recombinant sus scrofa trefoil factor 3 by Brevibacillus choshinensis

Wen

et al. 2020

Exp Ther Med

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Trefoil factor 3 (TFF3) is involved in cell adhesion, motility and apoptosis, regulates mucosal immunity and maintains the functional integrity of intestinal epithelia. The upregulation of TFF3 expression in the weaning rat intestine attracted our interest. The present study hypothesized that TFF3 may serve a role in preventing diarrhea in weaning piglets, which is an important consideration in the pig farming industry. Previous recombinant TFF3 protein expression yields obtained from Escherichia coli were too low and the bioactivity of the protein was poor. Hence, this expression system was unsuitable for industrial applications. The present study explored the production of recombinant sus scrofa TFF3 in a Brevibacillus choshinensis (B. choshinensis) expression system, aiming to enhance the expression level of bioactive protein. To achieve this, the sus scrofa TFF3-encoding gene fragment was fused into an E. coli-Brevibacillus shuttle vector pNCMO2. High levels of TFF3 (30 mg/l) were produced and secreted into the B. choshinensis culture medium in soluble form with a molecular mass of 13.6 kDa and high immunoreactivity in western blotting. Thus, Brevibacillus may be used to produce useful mucosal factors for biochemical analyses and mucosal protection, and in industrial applications to produce novel inhibitors of diarrhea.

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Secretory production of single-chain antibody (scFv) in Brevibacillus choshinensis using novel fusion partner

Cited by 19 publications

References 39 publications

Efficient extracellular expression of Bacillus deramificans pullulanase in Brevibacillus choshinensis

Efficient extracellular expression of Bacillus deramificans pullulanase in Brevibacillus choshinensis

Efficient Expression of Antibody Fragments with the Brevibacillus Expression System

Extracellular production of recombinant sus scrofa trefoil factor 3 by Brevibacillus choshinensis

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