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Expression of actively soluble antigen-binding fragment (Fab) antibody and GFP fused Fab in the cytoplasm of the engineered Escherichia coli

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Abstract

The expression of recombinant antibody fragments in the cytoplasmic space of Escherichia coli and the refolding process for restoring the structure and activity of such antibodies are not efficient. Herein, fragment antigen-binding (Fab) antibodies against miroestrol and deoxymiroestrol (MD-Fab) and their fusions with a green fluorescent protein (GFP) were expressed. The reactive MD-Fabs were successfully expressed as soluble and active forms in the cytoplasm of the SHuffle® T7 E. coli strain. Regarding the construct of MD-Fab alone, VH–CH1 could associate VL–CL into Fab in the oxidizing cytoplasm of the E. coli strain, and no additional in vitro refolding was needed. In the case of the fusions with GFP, when the C-terminus of VH–CH1 was linked with the N-terminus of GFP, the MD-Fab binding reactivity was retained, but the fluorescent activity of GFP interfered. When the C-terminus of GFP was linked to the N-terminus of VL–CL, the binding activity of MD-Fab was not observed. The constructed MD-Fabs had higher specificity toward deoxymiroestrol than the parental monoclonal antibody clone 12G11. In conclusion, MD-Fabs could be expressed using SHuffle® T7 E. coli cells. This process could be considered an economical, productive, and effective method to produce antibody fragments for immunoassay techniques.

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Acknowledgements

The authors would like to express gratitude to Dr. Chaiyo Chaichantipyuth, Faculty of Pharmaceutical Sciences, Chulalongkorn University, Thailand, for authentic standards.

Funding

This research was supported by the Faculty of Pharmaceutical Sciences, Graduate School of Khon Kaen University, Thailand (Grant Number 590H11), and the National Research Council of Thailand (NRCT). This research was partially supported by the new strategic research (P2P) project, Walailak University, Thailand.

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Authors and Affiliations

  1. Faculty of Pharmaceutical Sciences, Khon Kaen University, Khon Kaen, 40002, Thailand

    Supaluk Krittanai & Waraporn Putalun

  2. Research Group for Pharmaceutical Activities of Natural Products Using Pharmaceutical Biotechnology (PANPB), National Research University, Khon Kaen University, Khon Kaen, 40002, Thailand

    Supaluk Krittanai & Waraporn Putalun

  3. Graduate School of Pharmaceutical Sciences, Kyushu University, Fukuoka, 812-8582, Japan

    Seiichi Sakamoto

  4. Department of Pharmacognosy and Kampo, Faculty of Pharmaceutical Sciences, Sanyo-Onoda City University, 1-1-1 Daigaku-Dori, Sanyo-Onoda, Yamaguchi, 756-0884, Japan

    Hiroyuki Tanaka

  5. Faculty of Pharmaceutical Sciences, Ubon Ratchathani University, Ubon Ratchathani, 34190, Thailand

    Thaweesak Juengwatanatrakul

  6. School of Pharmacy, Walailak University, Thaiburi, Thasala, Nakhon Si Thammarat, 80160, Thailand

    Gorawit Yusakul

  7. Drug and Cosmetics Excellence Center, Walailak University, Thaiburi, Thasala, Nakhon Si Thammarat, 80160, Thailand

    Gorawit Yusakul

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  1. Supaluk Krittanai
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Correspondence to Gorawit Yusakul.

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Krittanai, S., Putalun, W., Sakamoto, S. et al. Expression of actively soluble antigen-binding fragment (Fab) antibody and GFP fused Fab in the cytoplasm of the engineered Escherichia coli. Mol Biol Rep 47, 4519–4529 (2020). https://doi.org/10.1007/s11033-020-05502-7

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