‘Zipbody’ leucine zipper-fused Fab in E. coli in vitro and in vivo expression systems
Abstract
A small antibody fragment, fragment of antigen binding (Fab), is favorable for various immunological assays. However, production efficiency of active Fab in microorganisms depends considerably on the clones. In this study, leucine zipper-peptide pairs that dimerize in parallel (ACID-p1 (LZA)/BASE-p1 (LZB) or c-Jun/c-Fos) were fused to the C-terminus of heavy chain (Hc, VH-CH1) and light chain (Lc, VL-CL), respectively, to accelerate the association of Hc and Lc to form Fab in Escherichia coli in vivo and in vitro expression systems. The leucine zipper-fused Fab named 'Zipbody' was constructed using anti-E. coli O157 monoclonal antibody obtained from mouse hybridoma and produced in both in vitro and in vivo expression systems in an active form, whereas Fab without the leucine zipper fusion was not. Similarly, Zipbody of rabbit monoclonal antibody produced in in vitro expression showed significant activity. The purified, mouse Zipbody produced in the E. coli strain Shuffle T7 Express had specificity toward the antigen; in bio-layer interferometry analysis, the KD value was measured to be 1.5-2.0 × 10(-8) M. These results indicate that leucine zipper fusion to Fab C-termini markedly enhances active Fab formation in E. coli.
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A Fab fragment bearing a leucine zipper pair at the C-termini of both heavy and light chains, named Zipbody, enhances chain pairing in the active form in E. coli cytoplasm expression and in cell-free protein synthesis systems.
“They also described an ad hoc engineered Fab fragment bearing a leucine zipper (LZ) pair at the C-termini of both the heavy and light chains, a construct named Zipbody. These extensions would enhance the chain pairing in the active form in both the E. coli cytoplasm expression and in the cell-free protein synthesis systems, a technology named Ecobody [124,125,126].”
From Evolution of Escherichia coli Expression System in Producing Antibody Recombinant Fragments (2020) · cites this paper as [126]
The Zipbody and SKIK peptide tag technologies have been developed to improve de novo synthesis of soluble and functional Fab fragments.
“The Zipbody and the SKIK peptide tag technologies have been developed to improve the de novo synthesis of soluble and functional Fab fragments [124,126].”
From Evolution of Escherichia coli Expression System in Producing Antibody Recombinant Fragments (2020) · cites this paper as [126]
Cites
Structure of the DNA-binding domains from NFAT, Fos and Jun bound specifically to DNA
Chen, L., Glover, J. N. M., Hogan, P. G., Rao, A., & Harrison, S. C. (1998) · Nature · doi:10.1038/32100 · Cited by 521 · see citing works
Efficient expression of full-length antibodies in the cytoplasm of engineered bacteria
Robinson, M.-P., Ke, N., Lobstein, J., Peterson, C., Szkodny, A., Mansell, T. J., Tuckey, C., Riggs, P. D., Colussi, P. A., Noren, C. J., et al. (2015) · Nature Communications · doi:10.1038/ncomms9072 · Cited by 131 · see citing works
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Evolution of Escherichia coli Expression System in Producing Antibody Recombinant Fragments
Sandomenico, A., Sivaccumar, J. P., & Ruvo, M. (2020) · International Journal of Molecular Sciences · doi:10.3390/ijms21176324 · Cited by 134 · see citing works
Rapid Generation of Monoclonal Antibodies from Single B Cells by Ecobody Technology
Ojima-Kato, T., Morishita, S., Uchida, Y., Nagai, S., Kojima, T., & Nakano, H. (2018) · Antibodies · doi:10.3390/antib7040038 · Cited by 23 · see citing works
Self-citation
- Authors
- Ojima-Kato, T., Fukui, K., Yamamoto, H., Hashimura, D., Miyake, S., Hirakawa, Y., Yamasaki, T., Kojima, T., & Nakano, H.
- Affiliations
- Nagoya University (JP)
- Advanced Science, Technology & Management Research Institute of Kyoto (JP)
- Corresponding author
- Nakano, Hideo
- Venue
- Protein Engineering Design and Selection
- Publisher
- Oxford University Press (OUP)
- Published
- 2016-02-21
- Volume
- 29
- Issue
- 4
- Pages
- 149-157
- ISSN
- 0269-2139 (ISSN-L)
- 1741-0126
- 1741-0134
- OpenAlex
- W2305060114
- Cited by
- 35
- References
- 2
- Citations by year
- 2026: 5 · 2025: 3 · 2024: 3 · 2023: 3 · 2022: 1 · 2021: 3 · 2020: 5 · 2019: 2 · 2018: 4 · 2017: 5 · 2016: 1
- FWCI
- 3.47
- Type
- journal article
- Language
- en
- Open access
- closed
- Locations
- Protein Engineering Design and Selection (publishedVersion)
- PubMed (publishedVersion)
- HighWire Press Open Archive (submittedVersion)
- Funders
- Aichi Science and Technology Foundation (Japan)
- Topics
- Health Sciences › Medicine › Radiology, Nuclear Medicine and Imaging › Monoclonal and Polyclonal Antibodies Research
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Protein purification and stability
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Biochemical and Structural Characterization
- Keywords
- leucine zipper, Fab fragment, antibody fragments, rabbit monoclonal antibodies, biolayer interferometry
- MeSH terms
- Animals, Cell-Free System, Escherichia coli, Immunoglobulin Fab Fragments, Rabbits, Recombinant Proteins, Protein Engineering, Leucine Zippers
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