Production of Single-Chain Fv Antibodies Specific for GA-Pyridine, an Advanced Glycation End-Product (AGE), with Reduced Inter-Domain Motion

被引：8

作者：

Fukuda, Natsuki ^{[1
]}

Noi, Kentaro ^{[2
,3
,10
]}

Weng, Lidong ^{[1
]}

Kobashigawa, Yoshihiro ^{[1
]}

Miyazaki, Hiromi ^{[1
]}

Wakeyama, Yukari ^{[1
]}

Takaki, Michiyo ^{[1
]}

Nakahara, Yusuke ^{[1
]}

Tatsuno, Yuka ^{[1
]}

Uchida-Kamekura, Makiyo ^{[1
,4
]}

Suwa, Yoshiaki ^{[1
]}

Sato, Takashi ^{[1
,13
]}

Ichikawa-Tomikawa, Naoki ^{[4
,11
]}

Nomizu, Motoyoshi ^{[4
,12
]}

Fujiwara, Yukio ^{[5
]}

Ohsaka, Fumina ^{[6
]}

Saito, Takashi ^{[6
]}

Maenaka, Katsumi ^{[6
]}

Kumeta, Hiroyuki ^{[7
]}

Shinya, Shoko ^{[8
]}

Kojima, Chojiro ^{[8
,9
]}

Ogura, Teru ^{[2
,3
]}

Morioka, Hiroshi ^{[1
]}

机构：

[1] Kumamoto Univ, Grad Sch Pharmaceut Sci, Dept Analyt & Biophys Chem, Chuo Ku, 5-1 Oe Honmachi, Kumamoto 8620973, Japan

[2] Kumamoto Univ, Dept Mol Cell Biol, Inst Mol Embryol & Genet, Chuo Ku, 2-2-1 Honjo, Kumamoto 8600811, Japan

[3] JST, CREST, 4-1-8 Honcho, Kawaguchi, Saitama 3320012, Japan

[4] Hokkaido Univ, Grad Sch Environm Earth Sci, Kita Ku, Kita 10 Nishi 5, Sapporo, Hokkaido 0600810, Japan

[5] Kumamoto Univ, Grad Sch Med Sci, Dept Cell Pathol, Chuo Ku, 1-1-1 Honjo, Kumamoto 8608556, Japan

[6] Hokkaido Univ, Grad Sch Pharmaceut Sci, Kita Ku, Kita 12 Nishi 6, Sapporo, Hokkaido 0600812, Japan

[7] Hokkaido Univ, Global Inst Collaborat Res & Educ, Global Stn Soft Matter, Kita Ku, Kita 15 Nishi 8, Sapporo, Hokkaido 0600815, Japan

[8] Osaka Univ, Inst Prot Res, Lab Mol Biophys, 3-2 Yamadaoka, Suita, Osaka 5650871, Japan

[9] Yokohama Natl Univ, Grad Sch Engn, Div Mat Sci & Chem Engn, Hodogaya Ku, 79-5 Tokiwadai, Yokohama, Kanagawa 2408501, Japan

[10] Osaka Univ, Grad Sch Engn Sci, Dept Mech Sci & Bioengn, 1-3 Machikaneyama, Toyonaka, Osaka 5608531, Japan

[11] Fukushima Med Univ, Sch Med, Dept Basic Pathol, 1 Hikariga Oka, Fukushima 9601295, Japan

[12] Tokyo Univ Pharm & Life Sci, Dept Clin Biochem, 1432-1 Horinouchi, Hachioji, Tokyo 1920392, Japan

[13] Hokkaido Pharmaceut Univ, Div Pharmaceut, Sch Pharm, 7-15-4-1 Maeda, Sapporo, Hokkaido 0068590, Japan

来源：

MOLECULES | 2017年 / 22卷 / 10期

基金：

日本学术振兴会; 日本科学技术振兴机构;

关键词：

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; SURFACE-PLASMON RESONANCE; BIOLOGICAL MACROMOLECULES; ESCHERICHIA-COLI; VARIABLE REGION; SCFV FRAGMENTS; BINDING-SITES; PHAGE DISPLAY; STABILITY; COMPLEX; DIMER;

D O I：

10.3390/molecules22101695

中图分类号：

Q5 [生物化学]; Q7 [分子生物学];

学科分类号：

071010 ; 081704 ;

摘要：

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 degrees 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-close dynamics of these scFvs by high-speed atomic force microscopy (HS-AFM), revealing that one of the compact clones was biased to the closed state. Finally, nuclear magnetic resonance (NMR) analysis revealed that peak intensity and line width became homogeneous, supporting that dynamic features and/or formation of oligomers was improved in the thus-obtained clone. These findings should contribute to the future industrial and therapeutic use of scFvs.

引用

页数：16

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