Optimization of culture conditions for stable expression of recombinant fc-fused human extracellular CD99 in HEK293T cells

• Published in: Protein expression and purification Vol. 200; p. 106151
• Main Authors: Thu, Myint Myat, Takheaw, Nuchjira, Laopajon, Witida, Pata, Supansa
• Format: Journal Article
• Language: English
• Published: Elsevier Inc 01.12.2022
• Subjects: CD99 counter receptor; Cytokine production; Mammalian cell expression system; Recombinant CD99 human IgG fusion protein; CD99 counter receptor; Cytokine production; Mammalian cell expression system; Recombinant CD99 human IgG fusion protein
• ISSN: 1046-5928; 1096-0279
• DOI: 10.1016/j.pep.2022.106151

CD99 has been demonstrated to play a key role in several biological processes, including the regulation of T-cell activation, cell adhesion, and cell migration. We have also demonstrated that CD99 and its ligands regulate proinflammatory cytokines in NK cells, monocytes and activated T cells. These data suggest CD99 as a potential therapeutic target in cancer. However, the molecular mechanisms by which CD99 and CD99 counter receptors participate in such processes are unclear. High-quality CD99 recombinant proteins produced in large amounts are essential for biological studies and clinical research. In this study, we optimized the various culture conditions for increasing amounts of recombinant protein production with good biological activity. Intracellular immunofluorescence staining was performed to identify the highly expressing CD99HIgG cells. We further investigated the culture conditions for recombinant protein production. A double antibody sandwich enzyme-linked immunosorbent assay was employed to determine the level of secreted CD99HIgG proteins in the culture supernatant of various culture conditions. Later, affinity chromatography using protein G was used to purify CD99HIgG proteins from the culture supernatant of three proper culture conditions. According to our previous report, which utilized Western blotting, the purified CD99HIgG obtained from all tested culture conditions is composed of the CD99 extracellular part fused with the human IgG Fc part in dimer form. For biological activity, the obtained CD99HIgG proteins showed the ability to ligate with the CD99 counter receptor, resulting in the induction of cytokine production. •High-quality CD99 recombinant proteins were produced in large amounts.•The culture conditions were evaluated for producing good biological activity CD99HIgG.•CD99HIgG proteins showed the ability to ligate with the CD99 counter receptor.