Chapter 5
The Intervening Removable Affinity Tag (iRAT) System
for the Production of Recombinant Antibody Fragments
Norimichi Nomura, Yayoi Nomura, Yumi Sato, and So Iwata
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.
Key words Recombinant antibody, Polyprotein, Secretory expression, Fv fragment, Fab fragment,
Crystallography, Genetically encoded fluorescent antibody, Therapeutic antibody, Biosimilar,
Immunodiagnostics
1 Introduction
Crystallography of biomedically relevant proteins often remains
stagnant because of challenges in preparing diffraction-quality crystals. In most cases, their molecular flexibility, conformational heterogeneity, or polydispersal characteristics in solution hinder
protein crystallization. Antibody-aided crystallography is a powerful strategy to manage this problem and can be applied to various
membrane proteins as well as heavily glycosylated and/or multidomain soluble proteins [1–7]. Fv and Fab antibody fragments
assist crystallization by increasing the hydrophilic surface area available for rigid crystal lattice formation. The bound antibody fragments reduce the inherent protein flexibility and conformational
heterogeneity, thereby increasing the chance of successful crystallization of difficult target proteins. In addition to crystallography,
Fab and Fv fragments have also been proven to be powerful as
Arnaud Poterszman (ed.), Multiprotein Complexes: Methods and Protocols, Methods in Molecular Biology, vol. 2247,
https://doi.org/10.1007/978-1-0716-1126-5_5, © Springer Science+Business Media, LLC, part of Springer Nature 2021
77
The Intervening Removable Affinity Tag (iRAT) System
for the Production of Recombinant Antibody Fragments
Norimichi Nomura, Yayoi Nomura, Yumi Sato, and So Iwata
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.
Key words Recombinant antibody, Polyprotein, Secretory expression, Fv fragment, Fab fragment,
Crystallography, Genetically encoded fluorescent antibody, Therapeutic antibody, Biosimilar,
Immunodiagnostics
1 Introduction
Crystallography of biomedically relevant proteins often remains
stagnant because of challenges in preparing diffraction-quality crystals. In most cases, their molecular flexibility, conformational heterogeneity, or polydispersal characteristics in solution hinder
protein crystallization. Antibody-aided crystallography is a powerful strategy to manage this problem and can be applied to various
membrane proteins as well as heavily glycosylated and/or multidomain soluble proteins [1–7]. Fv and Fab antibody fragments
assist crystallization by increasing the hydrophilic surface area available for rigid crystal lattice formation. The bound antibody fragments reduce the inherent protein flexibility and conformational
heterogeneity, thereby increasing the chance of successful crystallization of difficult target proteins. In addition to crystallography,
Fab and Fv fragments have also been proven to be powerful as
Arnaud Poterszman (ed.), Multiprotein Complexes: Methods and Protocols, Methods in Molecular Biology, vol. 2247,
https://doi.org/10.1007/978-1-0716-1126-5_5, © Springer Science+Business Media, LLC, part of Springer Nature 2021
77