Optimization of production and characterization of a recombinant soluble human Cripto-1 protein inhibiting self-renewal of cancer stem cells

Said M. Afify, Ghmkin Hassan, Hend M. Nawara, Maram H Zahra, Yanning Xu, Md Jahangir Alam, Koichi Saitoh, Hager Mansour, Hagar A. Abu Quora, Mona Sheta, Sadia Monzur, Juan Du, Sue Young Oh, Akimasa Seno, David S. Salomon, Masaharu Seno

研究成果査読

2 被引用数 (Scopus)

抄録

Human Cripto-1 is a member of the epidermal growth factor (EGF)-Cripto-FRL-1-Cryptic (CFC) family family and performs critical roles in cancer and various pathological and developmental processes. Recently we demonstrated that a soluble form of Cripto-1 suppresses the self-renewal and enhances the differentiation of cancer stem cells (CSCs). A functional form of soluble Cripto-1 was found to be difficult to obtain because of the 12 cysteine residues in the protein which impairs the folding process. Here, we optimized the protocol for a T7 expression system, purification from inclusion bodies under denatured conditions refolding of a His-tagged Cripto-1 protein. A concentrations of 0.2−0.4 mM isopropyl β-D-1-thiogalactopyranoside (IPTG) at 37°C was found to be the optimal concentration for Cripto-1 expression while imidazole at 0.5 M was the optimum concentration to elute the Cripto-1 protein from a Ni-column in the smallest volume. Cation exchange column chromatography of the Cripto-1 protein in the presence of 8 M urea exhibited sufficient elution profile at pH 5, which was more efficient at recovery. The recovery of the protein reached to more than 26.6% after refolding with arginine. The purified Cripto-1 exhibited high affinity to the anti-ALK-4 antibody and suppressed sphere forming ability of CSCs at high dose and induced cell differentiation.

本文言語English
ページ(範囲)1183-1196
ページ数14
ジャーナルJournal of Cellular Biochemistry
123
7
DOI
出版ステータスPublished - 7月 2022

ASJC Scopus subject areas

  • 生化学
  • 分子生物学
  • 細胞生物学

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