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

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Abstract

Halophilic β-lactamase (BLA) has been successfully used as a novel fusion partner for soluble expression of aggregation-prone foreign proteins in Escherichia coli cytoplasm (Appl Microbiol Biotechnol 86:649–658, 2010b). This halophilic BLA fusion technology was applied here for secretory expression in Brevibacillus. The “Brevibacillus in vivo cloning” method, recently developed by Higeta Shoyu group, for the construction and transformation of Brevibacillus expression vectors facilitates efficient screening of the production conditions of Brevibacillus expression system. Two single-chain antibodies (scFv), HyHEL-10 single chain scFv (scFvHEL) and anti-fluorescein single chain scFv (scFvFLU), were successfully secreted to culture supernatant as a fusion protein with halophilic BLA. The scFvHEL-His, purified after cleavage of BLA portion with thrombin, was fully active: it formed a stoichiometric complex with the antigen, lysozyme, and inhibited the enzymatic activity. The scFvFLU-His, similarly expressed and purified, stoichiometrically inhibited fluorescence intensity of fluorescein. The molecular mass of scFvHEL-His was determined to be 27,800 Da by light scattering measurements, indicating its monomeric structure in solution.

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Acknowledgments

We are grateful to Drs. Daisuke Ejima and Haruna Sato for the assistance of SEC MALS measurements. This work was supported by a Grant in Aid for Science Research (20580372, 23580475) to M.T. from MEXT Japan, and by funding from the Institute for Fermentation, Osaka to M.T.

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Authors and Affiliations

  1. Applied and Molecular Microbiology, Faculty of Agriculture, Kagoshima University, 1-21-24 Korimoto, Kagoshima, 890-0065, Japan

    Masao Tokunaga, Koji Yamasaki, Hiroko Tokunaga & Matsujiro Ishibashi

  2. Department of Research and Development, Higeta Shoyu Co., Ltd, 2-8 Chuo-cho, Choshi, Chiba, 288-8680, Japan

    Makoto Mizukami, Hiromasa Onishi, Hiroshi Hanagata & Akira Miyauchi

  3. Medical Proteomics Laboratory, Institute of Medical Science, The University of Tokyo, 4-6-1 Shirokanedai, Minato-ku, Tokyo, 108-8639, Japan

    Kouhei Tsumoto

  4. Alliance Protein Laboratories, 3957 Corte Cancion, Thousand Oaks, CA, 91360, USA

    Tsutomu Arakawa

Authors
  1. Masao Tokunaga
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  2. Makoto Mizukami
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  3. Koji Yamasaki
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  4. Hiroko Tokunaga
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  5. Hiromasa Onishi
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  6. Hiroshi Hanagata
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  7. Matsujiro Ishibashi
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  9. Kouhei Tsumoto
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  10. Tsutomu Arakawa
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Correspondence to Masao Tokunaga.

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Tokunaga, M., Mizukami, M., Yamasaki, K. et al. Secretory production of single-chain antibody (scFv) in Brevibacillus choshinensis using novel fusion partner. Appl Microbiol Biotechnol 97, 8569–8580 (2013). https://doi.org/10.1007/s00253-013-4695-2

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  • DOI: https://doi.org/10.1007/s00253-013-4695-2

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