NMR mapping of the highly flexible regions of C-13/N-15-labeled antibody TTAC-0001-Fab
- Cha, Soyoung; Lee, Weon Sup; Choi, Joonhyeok; Jeong, Jong Geun; Nam, Ju Ryoung; Kim, Jihong; Kim, Hak-Nam; Lee, Joon-Hwa; Yoo, Jin-San; Ryu, Kyoung-Seok
- Issue Date
- Antibody fab; Backbone chemical shift NMR assignment; E; coli periplasmic expression; Monoclonal antibody; NMR; TTAC-0001
- JOURNAL OF BIOMOLECULAR NMR, v.74, no.6-7, pp.311 - 319
- Journal Title
- JOURNAL OF BIOMOLECULAR NMR
- Start Page
- End Page
- Monoclonal antibody (mAb) drugs are clinically important for the treatment of various diseases. TTAC-0001 is under development as a new anti-cancer antibody drug targeting VEGFR-2. As the less severe toxicity of TTAC-0001 compared to Bevacizumab, likely due to the decreased in vivo half-life, seems to be related to its structural flexibility, it is important to map the exact flexible regions. Although the C-13/N-15-labeled protein is required for NMR analyses, it is difficult to obtain antibody fragments (Fab and scFv) containing disulfide bonds through general cytosolic expression in Escherichia coli (E. coli). Here, we notably increased the periplasmic expression of the C-13/N-15-labeled TTAC-0001-Fab (C-13/N-15-TTAC-Fab) through simple isopropyl beta-D-1-thiogalactopyranoside (IPTG)-induction at an increased optical density (1.5 OD600nm). Through NMR triple resonance experiments, two loop insertions (LI-1 between the V-H and C(H)1; LI-2 between the V-L and C-L) were confirmed to be highly flexible. The additional LIs could be another way to engineer the antibody by changing the pharmacokinetic properties.
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- 자연과학대학 > 화학과 > Journal Articles
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