7. Maintenance and management of insect cell culture can be
performed as described previously [16].
8. We usually add ~1.5 mL of P1 baculovirus stock per 1 L of Sf9
culture.
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).
References
1. Griffin L, Lawson A (2011) Antibody fragments as tools in crystallography. Clin Exp
Immunol 165:285–291
2. Lieberman RL, Culver JA, Entzminger KC
et al (2011) Crystallization chaperone strategies for membrane proteins. Methods
55:293–302
3. Hino T, Arakawa T, Iwanari H et al (2012) Gprotein-coupled receptor inactivation by an
allosteric inverse-agonist antibody. Nature
482:237–240
4. Nomura N, Verdon G, Kang HJ et al (2015)
Structure and mechanism of the mammalian
fructose
transporter
GLUT5.
Nature
526:397–401
5. Asada H, Horita S, Hirata K et al (2018) Crystal structure of the human angiotensin II type
2 receptor bound to an angiotensin II analog.
Nat Struct Mol Biol 25:570–576
6. Nagarathinam K, Nakada-Nakura Y, Parthier C
et al (2018) Outward open conformation of a
Major Facilitator Superfamily multidrug/H
+
antiporter provides insights into switching
mechanism. Nat Commun 9:4005
7. Toyoda Y, Morimoto K, Suno R et al (2019)
Ligand binding to human prostaglandin E
receptor EP4 at the lipid-bilayer interface. Nat
Chem Biol 15:18–26
8. Wu S, Avila-Sakar A, Kim J et al (2012) Fabs
enable single particle cryoEM studies of small
proteins. Structure 20:582–592
9. Koehl A, Hu H, Maeda S et al (2018) Structure
of the μ-opioid receptor-Gi protein complex.
Nature 558:547–552
10. Nomura Y, Sato Y, Suno R et al (2016) The
intervening removable affinity tag (iRAT) production system facilitates Fv antibody
fragment-mediated crystallography. Protein
Sci 25:2268–2276
11. Horita S, Nomura Y, Sato Y et al (2016) Highresolution crystal structure of the therapeutic
antibody pembrolizumab bound to the human
PD-1. Sci Rep 6:35297
12. Ono M, Horita S, Sato Y et al (2018) Structural
basis for tumor necrosis factor blockade with
the therapeutic antibody golimumab. Protein
Sci 27:1038–1046
13. Koerber JT, Hornsby MJ, Wells JA (2015) An
improved single-chain Fab platform for efficient display and recombinant expression. J
Mol Biol 427:576–586
14. Fields C, O’Connell D, Xiao S et al (2013)
Creation of recombinant antigen-binding
molecules derived from hybridomas secreting
specific antibodies. Nat Protoc 8:1125–1148
102
Norimichi Nomura et al.
performed as described previously [16].
8. We usually add ~1.5 mL of P1 baculovirus stock per 1 L of Sf9
culture.
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).
References
1. Griffin L, Lawson A (2011) Antibody fragments as tools in crystallography. Clin Exp
Immunol 165:285–291
2. Lieberman RL, Culver JA, Entzminger KC
et al (2011) Crystallization chaperone strategies for membrane proteins. Methods
55:293–302
3. Hino T, Arakawa T, Iwanari H et al (2012) Gprotein-coupled receptor inactivation by an
allosteric inverse-agonist antibody. Nature
482:237–240
4. Nomura N, Verdon G, Kang HJ et al (2015)
Structure and mechanism of the mammalian
fructose
transporter
GLUT5.
Nature
526:397–401
5. Asada H, Horita S, Hirata K et al (2018) Crystal structure of the human angiotensin II type
2 receptor bound to an angiotensin II analog.
Nat Struct Mol Biol 25:570–576
6. Nagarathinam K, Nakada-Nakura Y, Parthier C
et al (2018) Outward open conformation of a
Major Facilitator Superfamily multidrug/H
+
antiporter provides insights into switching
mechanism. Nat Commun 9:4005
7. Toyoda Y, Morimoto K, Suno R et al (2019)
Ligand binding to human prostaglandin E
receptor EP4 at the lipid-bilayer interface. Nat
Chem Biol 15:18–26
8. Wu S, Avila-Sakar A, Kim J et al (2012) Fabs
enable single particle cryoEM studies of small
proteins. Structure 20:582–592
9. Koehl A, Hu H, Maeda S et al (2018) Structure
of the μ-opioid receptor-Gi protein complex.
Nature 558:547–552
10. Nomura Y, Sato Y, Suno R et al (2016) The
intervening removable affinity tag (iRAT) production system facilitates Fv antibody
fragment-mediated crystallography. Protein
Sci 25:2268–2276
11. Horita S, Nomura Y, Sato Y et al (2016) Highresolution crystal structure of the therapeutic
antibody pembrolizumab bound to the human
PD-1. Sci Rep 6:35297
12. Ono M, Horita S, Sato Y et al (2018) Structural
basis for tumor necrosis factor blockade with
the therapeutic antibody golimumab. Protein
Sci 27:1038–1046
13. Koerber JT, Hornsby MJ, Wells JA (2015) An
improved single-chain Fab platform for efficient display and recombinant expression. J
Mol Biol 427:576–586
14. Fields C, O’Connell D, Xiao S et al (2013)
Creation of recombinant antigen-binding
molecules derived from hybridomas secreting
specific antibodies. Nat Protoc 8:1125–1148
102
Norimichi Nomura et al.