PDF(Pubmed)
Abstract：
UNASSIGNED: Anakinra must be injected daily due to its short half-life and this leads to lower patient compliance. Therefore, the aim of this study was to produce an interleukin-1 receptor antagonist (IL-1Ra) with albumin binding domain (ABD) as a novel fusion protein and evaluate its binding ability to albumin and its biological effects.
UNASSIGNED: The three-dimensional structure of IL-1Ra-ABD was predicted by MODELLER software and its interaction with IL-1R was evaluated by the HADDOCK server. The expression of IL-1Ra-ABD was performed in E. coli in fusion with intein 1 of pTWIN1 in soluble form and then purified. The affinity of IL-1Ra-ABD to human serum albumin (HSA) was determined on native-PAGE, and its release percent toward time was evaluated. Moreover, an MTT assay was used to determine the antagonizing properties of recombinant IL-1Ra-ABD against IL-1β in A375 and HEK293 cell lines.
UNASSIGNED: The stable complex of IL-1Ra-ABD with IL-1R established the absence of steric hindrance due to the addition of ABD to IL-1Ra. The expression induction of intein 1-IL-1Ra-ABD using 0.1 mM IPTG at 15 °C, and its cleavage represented bands approximately in 50 and 23 kDa. Furthermore, about 78% of IL-1Ra-ABD was attached to the HSA after 2 h of incubation, and the MTT assay showed no significant differences between the effects of IL-1Ra-ABD and native IL-1Ra in cell survival.
UNASSIGNED: The production of soluble IL-1Ra-ABD with no significant differences in IL-1Ra antagonizing effects was successfully performed. IL-1Ra-ABD showed suitable interaction with HSA and was released over time. However, the half-life of IL-1Ra-ABD in vivo must be determined in the subsequent investigations.
UNASSIGNED: The three-dimensional structure of IL-1Ra-ABD was predicted by MODELLER software and its interaction with IL-1R was evaluated by the HADDOCK server. The expression of IL-1Ra-ABD was performed in E. coli in fusion with intein 1 of pTWIN1 in soluble form and then purified. The affinity of IL-1Ra-ABD to human serum albumin (HSA) was determined on native-PAGE, and its release percent toward time was evaluated. Moreover, an MTT assay was used to determine the antagonizing properties of recombinant IL-1Ra-ABD against IL-1β in A375 and HEK293 cell lines.
UNASSIGNED: The stable complex of IL-1Ra-ABD with IL-1R established the absence of steric hindrance due to the addition of ABD to IL-1Ra. The expression induction of intein 1-IL-1Ra-ABD using 0.1 mM IPTG at 15 °C, and its cleavage represented bands approximately in 50 and 23 kDa. Furthermore, about 78% of IL-1Ra-ABD was attached to the HSA after 2 h of incubation, and the MTT assay showed no significant differences between the effects of IL-1Ra-ABD and native IL-1Ra in cell survival.
UNASSIGNED: The production of soluble IL-1Ra-ABD with no significant differences in IL-1Ra antagonizing effects was successfully performed. IL-1Ra-ABD showed suitable interaction with HSA and was released over time. However, the half-life of IL-1Ra-ABD in vivo must be determined in the subsequent investigations.
摘要：
■由于其半衰期短，必须每天注射Anakinra，这导致患者依从性降低。因此,本研究的目的是制备具有白蛋白结合域(ABD)的白细胞介素-1受体拮抗剂(IL-1Ra)作为新型融合蛋白,并评估其与白蛋白的结合能力及其生物学效应.
■通过MODELLER软件预测IL-1Ra-ABD的三维结构，并通过HADDOCK服务器评估其与IL-1R的相互作用。IL-1Ra-ABD的表达在与可溶形式的pTWIN1的内含肽1融合的大肠杆菌中进行,然后纯化。IL-1Ra-ABD对人血清白蛋白（HSA）的亲和力在天然-PAGE上测定，并评估了其随时间的释放百分比。此外，用MTT法测定重组IL-1Ra-ABD对A375和HEK293细胞系中IL-1β的拮抗特性。
■IL-1Ra-ABD与IL-1R的稳定复合物确立了由于将ABD添加到IL-1Ra中而不存在空间位阻。在15°C下使用0.1mMIPTG诱导内含肽1-IL-1Ra-ABD的表达，其切割代表约50和23kDa的条带。此外，孵育2小时后，约78％的IL-1Ra-ABD附着于HSA，MTT分析显示IL-1Ra-ABD和天然IL-1Ra在细胞存活中的作用之间没有显著差异。
■成功地产生了可溶性IL-1Ra-ABD，而IL-1Ra拮抗作用没有显着差异。IL-1Ra-ABD显示与HSA的合适相互作用,并随时间释放。然而,IL-1Ra-ABD在体内的半衰期必须在随后的研究中确定。
■通过MODELLER软件预测IL-1Ra-ABD的三维结构，并通过HADDOCK服务器评估其与IL-1R的相互作用。IL-1Ra-ABD的表达在与可溶形式的pTWIN1的内含肽1融合的大肠杆菌中进行,然后纯化。IL-1Ra-ABD对人血清白蛋白（HSA）的亲和力在天然-PAGE上测定，并评估了其随时间的释放百分比。此外，用MTT法测定重组IL-1Ra-ABD对A375和HEK293细胞系中IL-1β的拮抗特性。
■IL-1Ra-ABD与IL-1R的稳定复合物确立了由于将ABD添加到IL-1Ra中而不存在空间位阻。在15°C下使用0.1mMIPTG诱导内含肽1-IL-1Ra-ABD的表达，其切割代表约50和23kDa的条带。此外，孵育2小时后，约78％的IL-1Ra-ABD附着于HSA，MTT分析显示IL-1Ra-ABD和天然IL-1Ra在细胞存活中的作用之间没有显著差异。
■成功地产生了可溶性IL-1Ra-ABD，而IL-1Ra拮抗作用没有显着差异。IL-1Ra-ABD显示与HSA的合适相互作用,并随时间释放。然而,IL-1Ra-ABD在体内的半衰期必须在随后的研究中确定。
