糖工程干扰素的合理设计具有改进聚合状态的类似物:实验验证

M. Samoudi, Z. Minuchehr, S. Harcum, F. Tabandeh, N. Omid Yeganeh, M. Khodabandeh
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引用次数: 6

摘要

重组人干扰素;(rrhin -&bgr;)由于蛋白质聚集等不稳定性,临床使用的疗效低于预期。增加分子稳定性,糖工程已被用于改善临床疗效的治疗方法;然而,通常使用劳动密集的追踪和错误方法来识别额外的糖基化位点。在本研究中,两个rrhin -&bgr;通过理性硅法鉴定了具有一个额外糖基化位点L6T和S75N的类似物。这些rhIFN -&bgr;与中国仓鼠卵巢(CHO)密码子优化的天然人IFN-&bgr;(Opt-IFN-&bgr;),并使用相同的表达系统在CHO细胞中表达。观察到两种类似物的分子量均高于Opt-IFN-&bgr;,与高糖基化一致。体外生物实验显示高糖基化类似物和Opt-IFN-&bgr;有类似的活动。聚集研究表明,与Opt-IFN-&bgr;相比,这两种类似物的聚集倾向较低。这些实验研究验证了在保持生物功能的同时预测合适的糖基化位点的计算机策略。此外,这项工作描述了高糖基化的rrhin -&bgr;具有改善溶解度(即较低聚集)的类似物。这些发现,加上合理的硅设计,将使我们能够增加蛋白质糖基化,以提高治疗效果。
本文章由计算机程序翻译,如有差异,请以英文原文为准。
Rational design of glycoengineered interferon-&bgr; analogs with improved aggregation state: experimental validation
Recombinant human interferon-&bgr; (rhIFN-&bgr;) used clinically has lower efficacy than expected due to protein instabilities such as aggregation. Increasing molecular stability, glycoengineering has been used to improve clinical efficacy for a number of therapeutics; however, often labor-intensive trail-and-error approaches are used to identify additional glycosylation sites. In this study two rhIFN-&bgr; analogs with one additional glycosylation site, L6T and S75N, identified by a rational in silico approach, were characterized. These rhIFN-&bgr; analogs were synthesized in parallel with a Chinese hamster ovary (CHO) codon-optimized natural human IFN-&bgr; (Opt-IFN-&bgr;) and expressed in CHO cells using the same expression system. The molecular weights for both analogs were observed to be higher than Opt-IFN-&bgr;, consistent with hyper-glycosylation. The in vitro biological assay showed the hyper-glycosylated analogs and the Opt-IFN-&bgr; had similar activity. The aggregation studies demonstrated that both analogs had lower tendencies to aggregate compared to the Opt-IFN-&bgr;. These experimental studies validate the in silico strategy to predict suitable glycosylation sites that would be glycosylated, while maintaining biological function. Moreover, this work describes hyper-glycosylated rhIFN-&bgr; analogs with improved solubility (i.e. lower aggregation). These findings, together with the rational in silico design, will allow us to increase protein glycosylation with the goal to enhance therapeutic efficacy.
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