Abstract
Fv and Fab antibody fragments are versatile co-crystallization partners that aid in the structural determination of otherwise “uncrystallizable” proteins, including human/mammalian membrane proteins. Accessible methods for the rapid and reliable production of recombinant antibody fragments have been long sought. In this chapter, we describe the concept and protocols of the intervening removable affinity tag (iRAT) system for the efficient production of Fv and Fab fragments in milligram quantities, which are sufficient for structural studies. As an extension of the iRAT system, we also provide a new method for the creation of genetically encoded fluorescent Fab fragments, which are potentially useful as molecular devices in various basic biomedical and clinical procedures, such as immunofluorescence cytometry, bioimaging, and immunodiagnosis.
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Acknowledgments
We thank Shoichiro Horita, Hidetsugu Asada, and Tomoko Uemura for sharing their experiences on Sf9-baculovirus expression. The creation of the iRAT-based fluorescent Fab was conceived after being inspired by the pioneering work on multicolored fluorescent scFv by Markiv et al. [17]. This work was funded by the Strategic Basic Research Program from the Japan Science and Technology Agency (JST), the Basis for Supporting Innovative Drug Discovery and Life Science Research (BINDS) from the Japan Agency of Medical Research and Development (AMED), the Research on Development of New Drugs from the AMED, and the Grants-in-Aid for Scientific Research from the Japan Society for the Promotion of Science (JSPS) (Nos. 15K06968 and 18K05334).
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Nomura, N., Nomura, Y., Sato, Y., Iwata, S. (2021). The Intervening Removable Affinity Tag (iRAT) System for the Production of Recombinant Antibody Fragments. In: Poterszman, A. (eds) Multiprotein Complexes. Methods in Molecular Biology, vol 2247. Humana, New York, NY. https://doi.org/10.1007/978-1-0716-1126-5_5
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DOI: https://doi.org/10.1007/978-1-0716-1126-5_5
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