著者

Show Ishikawa Haruna Ishikawa Atsushi Sato

出版者

The Pharmaceutical Society of Japan

雑誌

Biological and Pharmaceutical Bulletin (ISSN:09186158)

巻号頁・発行日

vol.45, no.3, pp.284-291, 2022-03-01 (Released:2022-03-01)

参考文献数

27

被引用文献数

2

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Recently, we developed a platform strategy for hinge-deficient human immunoglobulin G1 (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 Escherichia (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, sodium dodecyl sulfate (SDS), 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate (CHAPS), or Triton X-100) in neutral (phosphate-buffered saline (PBS), pH 8) or alkaline (50 or 500 mM N-cyclohexyl-3-aminopropanesulfonic acid (CAPS), pH 11) buffer at 25 °C. Similar to the authentic CH2-CH3 produced in Chinese hamster ovary (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-polyacrylamide gel electrophoresis (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.

著者

三澤 久恵 佐口 清美 畠山 玲子 高尾 秀伸 竹田 恵子 阿部 大亮 島田 翔太郎 馬場 彪画 石川 晴菜 高山 眞帆 井上 奈南 岩崎 彩 Hisae Misawa Kiyomi Saguchi Reiko Hatakeyama Hidenobu Takao Keiko Takeda Daisuke Abe Shoutaro Shimada Hyouga Baba Haruna Ishikawa Maho Takayama Nanami Inoue Aya Iwasaki

出版者

神奈川工科大学

雑誌

神奈川工科大学研究報告. A・B, 人文社会科学編・理工学編 Research reports of Kanagawa Institute of Technology. 神奈川工科大学 編 (ISSN:21882878)

巻号頁・発行日

no.44, pp.25-30, 2020

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[Purpose] The purpose of this study was to analyze the characteristics of the elderly people who participated in the "group recminiscence therapy".[Method] The group reminiscence therapy was carried out for the elderly people of S area of A city from May to August 2018. The documents written by students who participated in the recollecting method as co-leaders was analyzed. The analytic theme was taken as "the meaning in which elderly people look back upon their life." The concepts was generated by qualitative research according to the Grounded Theory Approach (M-GTA).[Results] Altogether, 19 concepts were generated.The core concept was "perception about own existence realized through support of the surrounding people". The three core categories constituting the core concept were generated as "review of own life," "pride to self," "composing mind to the future."[Discussion] "Review of own life," like the flow in a river, makes one aware of the path traversed in life and proud of one's ability to handle events at the turning points in life with support from others, which in turn leads to "pride to self" "Things to accomplish in the future" relates to the approach to the end of life through cherishing the current way of living and building smooth relationships with others.