Molecules (Oct 2017)
Production of Single-Chain Fv Antibodies Specific for GA-Pyridine, an Advanced Glycation End-Product (AGE), with Reduced Inter-Domain Motion
- Natsuki Fukuda,
- Kentaro Noi,
- Lidong Weng,
- Yoshihiro Kobashigawa,
- Hiromi Miyazaki,
- Yukari Wakeyama,
- Michiyo Takaki,
- Yusuke Nakahara,
- Yuka Tatsuno,
- Makiyo Uchida-Kamekura,
- Yoshiaki Suwa,
- Takashi Sato,
- Naoki Ichikawa-Tomikawa,
- Motoyoshi Nomizu,
- Yukio Fujiwara,
- Fumina Ohsaka,
- Takashi Saitoh,
- Katsumi Maenaka,
- Hiroyuki Kumeta,
- Shoko Shinya,
- Chojiro Kojima,
- Teru Ogura,
- Hiroshi Morioka
Affiliations
- Natsuki Fukuda
- Department of Analytical and Biophysical Chemistry, Graduate School of Pharmaceutical Sciences, Kumamoto University, 5-1 Oe-honmachi, Chuo-ku, Kumamoto 862-0973, Japan
- Kentaro Noi
- Department of Molecular Cell Biology, Institute of Molecular Embryology and Genetics, Kumamoto University, 2-2-1 Honjo, Chuo-ku, Kumamoto 860-0811, Japan
- Lidong Weng
- Department of Analytical and Biophysical Chemistry, Graduate School of Pharmaceutical Sciences, Kumamoto University, 5-1 Oe-honmachi, Chuo-ku, Kumamoto 862-0973, Japan
- Yoshihiro Kobashigawa
- Department of Analytical and Biophysical Chemistry, Graduate School of Pharmaceutical Sciences, Kumamoto University, 5-1 Oe-honmachi, Chuo-ku, Kumamoto 862-0973, Japan
- Hiromi Miyazaki
- Department of Analytical and Biophysical Chemistry, Graduate School of Pharmaceutical Sciences, Kumamoto University, 5-1 Oe-honmachi, Chuo-ku, Kumamoto 862-0973, Japan
- Yukari Wakeyama
- Department of Analytical and Biophysical Chemistry, Graduate School of Pharmaceutical Sciences, Kumamoto University, 5-1 Oe-honmachi, Chuo-ku, Kumamoto 862-0973, Japan
- Michiyo Takaki
- Department of Analytical and Biophysical Chemistry, Graduate School of Pharmaceutical Sciences, Kumamoto University, 5-1 Oe-honmachi, Chuo-ku, Kumamoto 862-0973, Japan
- Yusuke Nakahara
- Department of Analytical and Biophysical Chemistry, Graduate School of Pharmaceutical Sciences, Kumamoto University, 5-1 Oe-honmachi, Chuo-ku, Kumamoto 862-0973, Japan
- Yuka Tatsuno
- Department of Analytical and Biophysical Chemistry, Graduate School of Pharmaceutical Sciences, Kumamoto University, 5-1 Oe-honmachi, Chuo-ku, Kumamoto 862-0973, Japan
- Makiyo Uchida-Kamekura
- Department of Analytical and Biophysical Chemistry, Graduate School of Pharmaceutical Sciences, Kumamoto University, 5-1 Oe-honmachi, Chuo-ku, Kumamoto 862-0973, Japan
- Yoshiaki Suwa
- Department of Analytical and Biophysical Chemistry, Graduate School of Pharmaceutical Sciences, Kumamoto University, 5-1 Oe-honmachi, Chuo-ku, Kumamoto 862-0973, Japan
- Takashi Sato
- Department of Analytical and Biophysical Chemistry, Graduate School of Pharmaceutical Sciences, Kumamoto University, 5-1 Oe-honmachi, Chuo-ku, Kumamoto 862-0973, Japan
- Naoki Ichikawa-Tomikawa
- Graduate School of Environmental Earth Science, Hokkaido University, Kita-10 Nishi-5, Kita-ku, Sapporo 060-0810, Japan
- Motoyoshi Nomizu
- Graduate School of Environmental Earth Science, Hokkaido University, Kita-10 Nishi-5, Kita-ku, Sapporo 060-0810, Japan
- Yukio Fujiwara
- Department of Cell Pathology, Graduate School of Medical Sciences, Kumamoto University, 1-1-1 Honjo, Chuo-ku, Kumamoto 860-8556, Japan
- Fumina Ohsaka
- Graduate School of Pharmaceutical Sciences, Hokkaido University, Kita-12 Nishi-6, Kita-ku, Sapporo 060-0812, Japan
- Takashi Saitoh
- Graduate School of Pharmaceutical Sciences, Hokkaido University, Kita-12 Nishi-6, Kita-ku, Sapporo 060-0812, Japan
- Katsumi Maenaka
- Graduate School of Pharmaceutical Sciences, Hokkaido University, Kita-12 Nishi-6, Kita-ku, Sapporo 060-0812, Japan
- Hiroyuki Kumeta
- Global Station of Soft Matter, Global Institution for Collaborative Research and Education, Hokkaido University, Kita-15 Nishi-8, Kita-ku, Sapporo 060-0815, Japan
- Shoko Shinya
- Laboratory of Molecular Biophysics, Institute for Protein Research, Osaka University, 3-2 Yamadaoka, Suita, Osaka 565-0871, Japan
- Chojiro Kojima
- Laboratory of Molecular Biophysics, Institute for Protein Research, Osaka University, 3-2 Yamadaoka, Suita, Osaka 565-0871, Japan
- Teru Ogura
- Department of Molecular Cell Biology, Institute of Molecular Embryology and Genetics, Kumamoto University, 2-2-1 Honjo, Chuo-ku, Kumamoto 860-0811, Japan
- Hiroshi Morioka
- Department of Analytical and Biophysical Chemistry, Graduate School of Pharmaceutical Sciences, Kumamoto University, 5-1 Oe-honmachi, Chuo-ku, Kumamoto 862-0973, Japan
- DOI
- https://doi.org/10.3390/molecules22101695
- Journal volume & issue
-
Vol. 22,
no. 10
p. 1695
Abstract
Due to their lower production cost compared with monoclonal antibodies, single-chain variable fragments (scFvs) have potential for use in several applications, such as for diagnosis and treatment of a range of diseases, and as sensor elements. However, the usefulness of scFvs is limited by inhomogeneity through the formation of dimers, trimers, and larger oligomers. The scFv protein is assumed to be in equilibrium between the closed and open states formed by assembly or disassembly of VH and VL domains. Therefore, the production of an scFv with equilibrium biased to the closed state would be critical to overcome the problem in inhomogeneity of scFv for industrial or therapeutic applications. In this study, we obtained scFv clones stable against GA-pyridine, an advanced glycation end-product (AGE), by using a combination of a phage display system and random mutagenesis. Executing the bio-panning at 37 °C markedly improved the stability of scFvs. We further evaluated the radius of gyration by small-angle X-ray scattering (SAXS), obtained compact clones, and also visualized open
Keywords
- GA-pyridine
- single-chain variable fragment
- phage display
- isothermal titration calorimetry
- differential scanning calorimetry
- small-angle X-ray scattering
- high-speed atomic force microscopy
- NMR analysis
- inter-domain motion
