Production of Single-Chain Fv Antibodies Specific for GA-Pyridine, an Advanced Glycation End-Product (AGE), with Reduced Inter-Domain Motion
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 inhomogen...
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| Format: | Article |
| Language: | English |
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MDPI AG
2017-10-01
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| Series: | Molecules |
| Subjects: | |
| Online Access: | https://www.mdpi.com/1420-3049/22/10/1695 |
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doaj-ec91204768074e62bae41965348e2ac8 |
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| record_format |
Article |
| collection |
DOAJ |
| language |
English |
| format |
Article |
| sources |
DOAJ |
| author |
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 |
| spellingShingle |
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 Production of Single-Chain Fv Antibodies Specific for GA-Pyridine, an Advanced Glycation End-Product (AGE), with Reduced Inter-Domain Motion Molecules 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 |
| author_facet |
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 |
| author_sort |
Natsuki Fukuda |
| title |
Production of Single-Chain Fv Antibodies Specific for GA-Pyridine, an Advanced Glycation End-Product (AGE), with Reduced Inter-Domain Motion |
| title_short |
Production of Single-Chain Fv Antibodies Specific for GA-Pyridine, an Advanced Glycation End-Product (AGE), with Reduced Inter-Domain Motion |
| title_full |
Production of Single-Chain Fv Antibodies Specific for GA-Pyridine, an Advanced Glycation End-Product (AGE), with Reduced Inter-Domain Motion |
| title_fullStr |
Production of Single-Chain Fv Antibodies Specific for GA-Pyridine, an Advanced Glycation End-Product (AGE), with Reduced Inter-Domain Motion |
| title_full_unstemmed |
Production of Single-Chain Fv Antibodies Specific for GA-Pyridine, an Advanced Glycation End-Product (AGE), with Reduced Inter-Domain Motion |
| title_sort |
production of single-chain fv antibodies specific for ga-pyridine, an advanced glycation end-product (age), with reduced inter-domain motion |
| publisher |
MDPI AG |
| series |
Molecules |
| issn |
1420-3049 |
| publishDate |
2017-10-01 |
| description |
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 |
| topic |
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 |
| url |
https://www.mdpi.com/1420-3049/22/10/1695 |
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doaj-ec91204768074e62bae41965348e2ac82020-11-24T23:55:27ZengMDPI AGMolecules1420-30492017-10-012210169510.3390/molecules22101695molecules22101695Production of Single-Chain Fv Antibodies Specific for GA-Pyridine, an Advanced Glycation End-Product (AGE), with Reduced Inter-Domain MotionNatsuki Fukuda0Kentaro Noi1Lidong Weng2Yoshihiro Kobashigawa3Hiromi Miyazaki4Yukari Wakeyama5Michiyo Takaki6Yusuke Nakahara7Yuka Tatsuno8Makiyo Uchida-Kamekura9Yoshiaki Suwa10Takashi Sato11Naoki Ichikawa-Tomikawa12Motoyoshi Nomizu13Yukio Fujiwara14Fumina Ohsaka15Takashi Saitoh16Katsumi Maenaka17Hiroyuki Kumeta18Shoko Shinya19Chojiro Kojima20Teru Ogura21Hiroshi Morioka22Department of Analytical and Biophysical Chemistry, Graduate School of Pharmaceutical Sciences, Kumamoto University, 5-1 Oe-honmachi, Chuo-ku, Kumamoto 862-0973, JapanDepartment of Molecular Cell Biology, Institute of Molecular Embryology and Genetics, Kumamoto University, 2-2-1 Honjo, Chuo-ku, Kumamoto 860-0811, JapanDepartment of Analytical and Biophysical Chemistry, Graduate School of Pharmaceutical Sciences, Kumamoto University, 5-1 Oe-honmachi, Chuo-ku, Kumamoto 862-0973, JapanDepartment of Analytical and Biophysical Chemistry, Graduate School of Pharmaceutical Sciences, Kumamoto University, 5-1 Oe-honmachi, Chuo-ku, Kumamoto 862-0973, JapanDepartment of Analytical and Biophysical Chemistry, Graduate School of Pharmaceutical Sciences, Kumamoto University, 5-1 Oe-honmachi, Chuo-ku, Kumamoto 862-0973, JapanDepartment of Analytical and Biophysical Chemistry, Graduate School of Pharmaceutical Sciences, Kumamoto University, 5-1 Oe-honmachi, Chuo-ku, Kumamoto 862-0973, JapanDepartment of Analytical and Biophysical Chemistry, Graduate School of Pharmaceutical Sciences, Kumamoto University, 5-1 Oe-honmachi, Chuo-ku, Kumamoto 862-0973, JapanDepartment of Analytical and Biophysical Chemistry, Graduate School of Pharmaceutical Sciences, Kumamoto University, 5-1 Oe-honmachi, Chuo-ku, Kumamoto 862-0973, JapanDepartment of Analytical and Biophysical Chemistry, Graduate School of Pharmaceutical Sciences, Kumamoto University, 5-1 Oe-honmachi, Chuo-ku, Kumamoto 862-0973, JapanDepartment of Analytical and Biophysical Chemistry, Graduate School of Pharmaceutical Sciences, Kumamoto University, 5-1 Oe-honmachi, Chuo-ku, Kumamoto 862-0973, JapanDepartment of Analytical and Biophysical Chemistry, Graduate School of Pharmaceutical Sciences, Kumamoto University, 5-1 Oe-honmachi, Chuo-ku, Kumamoto 862-0973, JapanDepartment of Analytical and Biophysical Chemistry, Graduate School of Pharmaceutical Sciences, Kumamoto University, 5-1 Oe-honmachi, Chuo-ku, Kumamoto 862-0973, JapanGraduate School of Environmental Earth Science, Hokkaido University, Kita-10 Nishi-5, Kita-ku, Sapporo 060-0810, JapanGraduate School of Environmental Earth Science, Hokkaido University, Kita-10 Nishi-5, Kita-ku, Sapporo 060-0810, JapanDepartment of Cell Pathology, Graduate School of Medical Sciences, Kumamoto University, 1-1-1 Honjo, Chuo-ku, Kumamoto 860-8556, JapanGraduate School of Pharmaceutical Sciences, Hokkaido University, Kita-12 Nishi-6, Kita-ku, Sapporo 060-0812, JapanGraduate School of Pharmaceutical Sciences, Hokkaido University, Kita-12 Nishi-6, Kita-ku, Sapporo 060-0812, JapanGraduate School of Pharmaceutical Sciences, Hokkaido University, Kita-12 Nishi-6, Kita-ku, Sapporo 060-0812, JapanGlobal Station of Soft Matter, Global Institution for Collaborative Research and Education, Hokkaido University, Kita-15 Nishi-8, Kita-ku, Sapporo 060-0815, JapanLaboratory of Molecular Biophysics, Institute for Protein Research, Osaka University, 3-2 Yamadaoka, Suita, Osaka 565-0871, JapanLaboratory of Molecular Biophysics, Institute for Protein Research, Osaka University, 3-2 Yamadaoka, Suita, Osaka 565-0871, JapanDepartment of Molecular Cell Biology, Institute of Molecular Embryology and Genetics, Kumamoto University, 2-2-1 Honjo, Chuo-ku, Kumamoto 860-0811, JapanDepartment of Analytical and Biophysical Chemistry, Graduate School of Pharmaceutical Sciences, Kumamoto University, 5-1 Oe-honmachi, Chuo-ku, Kumamoto 862-0973, JapanDue 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 openhttps://www.mdpi.com/1420-3049/22/10/1695GA-pyridinesingle-chain variable fragmentphage displayisothermal titration calorimetrydifferential scanning calorimetrysmall-angle X-ray scatteringhigh-speed atomic force microscopyNMR analysisinter-domain motion |