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Updated: Nov 26, 2025

Orthogonal Protein Purification Facilitated by a Small Bispecific Affinity Tag
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The Intervening Removable Affinity Tag (iRAT) System for the Production of Recombinant Antibody Fragments

Norimichi Nomura1, Yayoi Nomura2, Yumi Sato2

  • 1Department of Cell Biology, Graduate School of Medicine, Kyoto University, Kyoto, Japan. nnomura@mfour.med.kyoto-u.ac.jp.

Methods in Molecular Biology (Clifton, N.J.)
|December 10, 2020
PubMed

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.

Keywords:
BiosimilarCrystallographyFab fragmentFv fragmentGenetically encoded fluorescent antibodyImmunodiagnosticsPolyproteinRecombinant antibodySecretory expressionTherapeutic antibody

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