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Efficient heterologous expression and secretion in Aspergillus oryzae of a llama variable heavy-chain antibody fragment VHH against EGFR

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Abstract

We have constructed a filamentous fungus Aspergillus oryzae that secretes a llama variable heavy-chain antibody fragment (VHH) that binds specifically to epidermal growth factor receptor (EGFR) in a culture medium. A major improvement in yield was achieved by fusing the VHH with a Taka-amylase A signal sequence (sTAA) and a segment of 28 amino acids from the N-terminal region of Rhizopus oryzae lipase (N28). The yields of secreted, immunologically active anti-EGFR VHH reached 73.8 mg/1 in a Sakaguchi flask. The VHH fragments were released from the sTAA or N28 proteins by an indigenous A. oryzae protease during cultivation. The purified recombinant VHH fragment was specifically recognized and could bind to the EGFR with a high affinity.

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Acknowledgments

The A. oryzae niaD mutant (strain IF4) was kindly provided by Research Institute, Gekkeikan Sake Co., Kyoto, Japan. Nanami Nakashima is thanked for technical support and useful discussion. Sachiko Nakamura-Tsuruta and Tomokazu Amino are thanked for binding kinetics analysis by surface plasmon resonance. Satoshi Kawamoto, Research Center for Environmental Genomics, Kobe University, is thanked for protein sequencing analysis. This work was partially supported by Special Coordination Funds for Promoting Science and Technology, Creation of Innovation Centers for Advanced Interdisciplinary Research Areas (Innovative Bioproduction, Kobe), Ministry of Education, Culture, Sports, Science and Technology, Japan.

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Authors and Affiliations

  1. Organization of Advanced Science and Technology, Kobe University, 1-1 Rokkodai, Nada, Kobe, 657-8501, Japan

    Fumiyoshi Okazaki

  2. Department of Chemical Science and Engineering, Graduate School of Engineering, Kobe University, 1-1 Rokkodai, Nada, Kobe, 657-8501, Japan

    Jun-ichi Aoki, Soichiro Tabuchi, Tsutomu Tanaka, Chiaki Ogino & Akihiko Kondo

Authors
  1. Fumiyoshi Okazaki
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  2. Jun-ichi Aoki
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  3. Soichiro Tabuchi
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  4. Tsutomu Tanaka
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  5. Chiaki Ogino
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  6. Akihiko Kondo
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Corresponding author

Correspondence to Akihiko Kondo.

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Fumiyoshi Okazaki and Jun-ichi Aoki contributed equally to this work.

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Okazaki, F., Aoki, Ji., Tabuchi, S. et al. Efficient heterologous expression and secretion in Aspergillus oryzae of a llama variable heavy-chain antibody fragment VHH against EGFR. Appl Microbiol Biotechnol 96, 81–88 (2012). https://doi.org/10.1007/s00253-012-4158-1

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