サトウ　アツシ   佐藤　淳    所属   応用生物学部 応用生物学科    職種   教授
言語種別	英語
発行・発表の年月	2022/03
形態種別	学術論文
査読	査読あり
標題	Improved refolding of a human IgG1 Fc (CH2-CH3) scaffold from its inclusion body in E. coli by alkaline solubilization
執筆形態	共著
掲載誌名	Biological & Pharmaceutical Bulletin
掲載区分	国外
出版社・発行元	The Pharmaceutical Society of Japan
巻・号・頁	45,pp.284-291
担当区分	責任著者
著者・共著者	Show Ishikawa , Haruna Ishikawa, and Atsushi Sato
概要	Recently, we developed a platform strategy for hinge-deficient human IgG1 Fc fusion as a non-immunostimulatory Fc fusion system. As a starting point to establish a promising approach for generating hinge-deficient Fc fusion proteins in E. coli, we selected a CH2-CH3 scaffold as a model protein for evaluation. Recombinant CH2-CH3, expressed as inclusion bodies, was solubilized with various denaturants (urea, sarkosyl, SDS, CHAPS, or Triton X-100) in neutral (PBS, pH 8) or alkaline (50 mM or 500 mM CAPS, pH 11) buffer at 25 °C. Similar to the authentic CH2-CH3 produced in CHO cells, all denaturants, except urea in CAPS buffer but not in PBS, were found to elicit the dimer formation of solubilized CH2-CH3 on SDS-PAGE. After dialysis with PBS, sarkosyl-soluble CH2-CH3 inclusion bodies were successfully purified using protein G-Sepharose, indicating their successful refolding. Compared to the purified CH2-CH3 from its sarkosyl-soluble inclusion bodies in neutral buffer, that in 500 mM CAPS alkaline buffer revealed substantial structure-related similarities, such as secondary structures and thermal stabilities, as measured by circular dichroism spectroscopy, to authentic CH2-CH3. Native PAGE analysis also supported the above data. Therefore, solubilization at alkaline pH is an essential factor that promotes the refolding of CH2-CH3. Dimer formation of CH2-CH3 on SDS-PAGE may act as a surrogate marker for its protein refolding status. Our observations may provide important hints toward downstream processing of Fc-fusion production in E.coli.
外部リンクURL	https://www.jstage.jst.go.jp/article/bpb/45/3/45_b21-00796/_article/-char/ja/