title: A novel bifunctional protein TNFR2-Fc-IL-1ra (TFI): expression, purification and its neutralization activity of inflammatory factors.
abstract: Tumor necrosis factor receptor (TNF) and internleukin-1 (IL-1) are the most potent proinflammatory cytokines involving in autoimmune and inflammatory human diseases. Many anti-inflammatory agents have been exploited for anti-inflammation treatments by targeting cytokines including TNF and IL-1. Theoretically, simultaneously neutralizing or blocking two important inflammatory mediators may achieve a synergistic therapeutic effect. We have developed a recombinant fusion protein, TNFR2-Fc-IL-1ra (TFI), which consists of a TNF-neutralizing domain that specifically binds to TNF-α, an IL-1 receptor antagonist domain, and a dimerization Fc portion of human IgG1, for bifunctional inflammatory inhibitor. Recombinant DNA expressing the sequence of this fusion protein was expressed in CHO-S cells. The protein product was purified using a two-step purification protocol and the identity of the protein was confirmed by western blot analysis. The purified recombinant protein had a purity of about 98 % as determined by HPLC, and a molecular mass of 164.6 kDa as determined by matrix-assisted laser desorption/ionization-time of flight mass spectrometry. The results of cell binding inhibition indicate that TFI was able to strongly neutralize TNF activity and antagonize IL-1r activity, suggesting that TFI may be used as a bifunctional ligand with enhanced anti-inflammatory effect. The result obtained in this study may provide a platform for extending bifunctional anti-inflammatory drug development.
authors.affiliation: Institute of Process Engineering, Chinese Academy of Sciences, Beijing, China. bxie@mail.ipe.ac.cn
authors.forename: Bo
authors.initials: B
authors.lastname: Xie
authors.forename: Shan
authors.initials: S
authors.lastname: Liu
authors.forename: Shuyin
authors.initials: S
authors.lastname: Wu
authors.forename: Alan
authors.initials: A
authors.lastname: Chang
authors.forename: Weiyuan
authors.initials: W
authors.lastname: Jin
authors.forename: Zhixiang
authors.initials: Z
authors.lastname: Guo
authors.forename: Shuaidong
authors.initials: S
authors.lastname: Ye
authors.forename: Mizhou
authors.initials: M
authors.lastname: Hui
mesh_terms: D000818:Animals; D016466:CHO Cells; D002460:Cell Line; D006224:Cricetinae; D003412:Cricetulus; D006801:Humans; D007074:Immunoglobulin G / Q000235:genetics / Q000378:metabolism; D007249:Inflammation / Q000235:genetics* / Q000378:metabolism; D053590:Interleukin 1 Receptor Antagonist Protein / Q000235:genetics* / Q000378:metabolism; D051379:Mice; D017472:Receptors, Interleukin-1 / Q000235:genetics / Q000378:metabolism; D047889:Receptors, Tumor Necrosis Factor, Type II / Q000235:genetics* / Q000378:metabolism; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D014409:Tumor Necrosis Factor-alpha / Q000235:genetics / Q000378:metabolism
chemical_list: D007074:Immunoglobulin G; D053590:Interleukin 1 Receptor Antagonist Protein; D017472:Receptors, Interleukin-1; D047889:Receptors, Tumor Necrosis Factor, Type II; D011994:Recombinant Proteins; D014409:Tumor Necrosis Factor-alpha