Highly efficient production of VHH antibody fragments in Brevibacillus choshinensis expression system

Mizukami, Masaaki; Tokunaga, Hiroko; Onishi, Hiromasa; Ueno, Yoshimitsu; Hanagata, Hiroshi; Miyazaki, Nobuo; Kiyose, Norihiko; Ito, Yuji; Ishibashi, Matsujiro; Hagihara, Yoshihisa; Arakawa, Tsutomu; Miyauchi, Akira; Tokunaga, Masao

doi:10.1016/j.pep.2014.09.017

Cited by 36 publications

(17 citation statements)

References 40 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: 96%

“…The Brevibacillus choshinensis (formerly Bacillus brevis) expression system has been developed for highly efficient production of heterologous proteins, including cellulase [18], metalloproteinase [23], and VHH antibody [22]. Brevibacillus choshinensis, a gram-positive bacterium, has exceptional capacity for extracellular protein secretion that is particularly suitable for the production of secretory proteins [15,21,25].…”

Section: Introductionmentioning

confidence: 99%

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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: 96%

Section: Introductionmentioning

confidence: 99%

Efficient extracellular expression of Bacillus deramificans pullulanase in Brevibacillus choshinensis

Zou

Duan

2016

Journal of Industrial Microbiology and Biotechnology

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“…Thus, it is important to use genetic engineering strategies to produce large amounts of recombinant TFF3 for biochemical and biomedical applications. B. choshinensis has an exceptional capacity to produce heterologous proteins (23,24,(33)(34)(35). Target proteins can be produced and secreted efficiently with a high yield by Brevibacillus expression systems (33).…”

Section: Discussionmentioning

confidence: 99%

“…Target proteins can be produced and secreted efficiently with a high yield by Brevibacillus expression systems (33). The Brevibacillus expression system is suitable for producing eukaryotic proteins (23,24,33,34). Compared with traditional E. coli expression systems, the genus Brevibacillus, including thermophilic, alkalophilic, psychrophilic, acidophilic and halophilic strains, use more carbon for heterotrophic or autotrophic growth (34).…”

Section: Discussionmentioning

confidence: 99%

“…The Brevibacillus expression system is suitable for producing eukaryotic proteins (23,24,33,34). Compared with traditional E. coli expression systems, the genus Brevibacillus, including thermophilic, alkalophilic, psychrophilic, acidophilic and halophilic strains, use more carbon for heterotrophic or autotrophic growth (34). Brevibacillus expression systems can produce certain secretory or cytoplasmic proteins that E. coli expression systems fail to produce (35).…”

Section: Discussionmentioning

confidence: 99%

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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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Simple production of resilin‐like protein hydrogels using the Brevibacillus secretory expression system and column‐free purification

Homma

Terui

Yokoyama

et al. 2022

Biotech & Bioengineering

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Resilin, an insect structural protein, has excellent flexibility, photocrosslinking properties, and temperature responsiveness. Recombinant resilin‐like proteins (RLPs) can be fabricated into three‐dimensional (3D) structures for use as cell culture substrates and highly elastic materials. A simplified, high‐yielding production process for RLPs is required for their widespread application. This study proposes a simple production process combining extracellular expression using Brevibacillus choshinensis (B. choshinensis) and rapid column‐free purification. Extracellular production was tested using four representative signal peptides; B. choshinensis was found to efficiently secrete Rec1, an RLP derived from Drosophila melanogaster, regardless of the type of signal peptide. However, it was suggested that Rec1 is altered by an increase in the pH of the culture medium associated with prolonged incubation. Production in a jar fermentor with controllable pH yielded 530 mg Rec1 per liter of culture medium, which is superior to productivity using other hosts. The secreted Rec1 was purified from the culture supernatant via (NH4)2SO4 and ethanol precipitations, and the purified Rec1 was applied to ring‐shaped 3D hydrogels. These results indicate that the combination of secretory production using B. choshinensis and column‐free purification can accelerate the further application of RLPs.

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Highly efficient production of VHH antibody fragments in Brevibacillus choshinensis expression system

Cited by 36 publications

References 40 publications

Efficient extracellular expression of Bacillus deramificans pullulanase in Brevibacillus choshinensis

Efficient extracellular expression of Bacillus deramificans pullulanase in Brevibacillus choshinensis

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

Simple production of resilin‐like protein hydrogels using the Brevibacillus secretory expression system and column‐free purification

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