The intervening removable affinity tag (iRAT) production system facilitates Fv antibody fragment-mediated crystallography.
Fv antibody fragments have been used as co-crystallization partners in structural biology, particularly in membrane protein crystallography. However, there are inherent technical issues associated with the large-scale production of soluble, functional Fv fragments through conventional methods in various expression systems. To circumvent these problems, we developed a new method, in which a single synthetic polyprotein consisting of a variable light (VL) domain, an intervening removable affinity tag (iRAT), and a variable heavy (VH) domain is expressed by a Gram-positive bacterial secretion system. This method ensures stoichiometric expression of VLand VHfrom the monocistronic construct followed by proper folding and assembly of the two variable domains. The iRAT segment can be removed by a site-specific protease during the purification process to yield tag-free Fv fragments suitable for crystallization trials. In vitro refolding step is not required to obtain correctly folded Fv fragments. As a proof of concept, we tested the iRAT-based production of multiple Fv fragments, including a crystallization chaperone for a mammalian membrane protein as well as FDA-approved therapeutic antibodies. The resulting Fv fragments were functionally active and crystallized in complex with the target proteins. The iRAT system is a reliable, rapid and broadly applicable means of producing milligram quantities of Fv fragments for structural and biochemical studies.
Department of Cell Biology, Graduate School of Medicine, Kyoto University, Yoshida-Konoe-cho, Sakyo-ku, Kyoto, 606-8501, Japan.|Japan Science and Technology Agency, Research Acceleration Program, Membrane Protein Crystallography Project, Yoshida-Konoe-cho, Sakyo-ku, Kyoto, 606-8501, Japan.
Yayoi
0000-0002-6330-2239
Y
Nomura
Department of Cell Biology, Graduate School of Medicine, Kyoto University, Yoshida-Konoe-cho, Sakyo-ku, Kyoto, 606-8501, Japan.|Japan Science and Technology Agency, Research Acceleration Program, Membrane Protein Crystallography Project, Yoshida-Konoe-cho, Sakyo-ku, Kyoto, 606-8501, Japan.
Yumi
Y
Sato
Department of Cell Biology, Graduate School of Medicine, Kyoto University, Yoshida-Konoe-cho, Sakyo-ku, Kyoto, 606-8501, Japan.
Ryoji
R
Suno
Department of Cell Biology, Graduate School of Medicine, Kyoto University, Yoshida-Konoe-cho, Sakyo-ku, Kyoto, 606-8501, Japan.
Shoichiro
S
Horita
Department of Cell Biology, Graduate School of Medicine, Kyoto University, Yoshida-Konoe-cho, Sakyo-ku, Kyoto, 606-8501, Japan.|Japan Science and Technology Agency, Research Acceleration Program, Membrane Protein Crystallography Project, Yoshida-Konoe-cho, Sakyo-ku, Kyoto, 606-8501, Japan.|RIKEN SPring-8 Center, Kouto, Sayo-cho, Sayo-gun, Hyogo, 679-5148, Japan.
So
S
Iwata
Department of Cell Biology, Graduate School of Medicine, Kyoto University, Yoshida-Konoe-cho, Sakyo-ku, Kyoto, 606-8501, Japan.|Japan Science and Technology Agency, Research Acceleration Program, Membrane Protein Crystallography Project, Yoshida-Konoe-cho, Sakyo-ku, Kyoto, 606-8501, Japan.
Norimichi
N
Nomura
D058675:Brevibacillus / Q000235:genetics / Q000378:metabolism*; D018360:Crystallography, X-Ray / Q000379:methods; D006801:Humans; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification; D057127:Single-Chain Antibodies / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification
D011993:Recombinant Fusion Proteins; D057127:Single-Chain Antibodies
Fv fragment; Gram-positive bacteria; antibody; crystallization chaperone; membrane protein; polyprotein; secretory expression; therapeutic antibody