One-step production of bioactive proteins through simultaneous PEGylation and refolding.
One-step production of bioactive proteins through simultaneous PEGylation and refolding.
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DOI：
10.1021/bc400314f
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发表时间：
2014-01
影响因子：
4.7
通讯作者：
Jianlou Niu;Yanlin Zhu;Lintao Song;Yaoyao Xie;Yi Zhang;Huiyan Wang;Xiaokun Li;Bailin Liu;L. Cai;Zhifeng Huang
中科院分区：
文献类型：
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作者：
Jianlou Niu;Yanlin Zhu;Lintao Song;Yaoyao Xie;Yi Zhang;Huiyan Wang;Xiaokun Li;Bailin Liu;L. Cai;Zhifeng Huang
蛋白质治疗药物的生产通常涉及细菌包涵体的体外复性和随后的聚乙二醇化，以提高蛋白质的稳定性和血浆半衰期。在这里，我们设计了一种新的策略，通过结合聚乙二醇化和蛋白质复性(IPPR)一步生产具有良好生物活性和提高生物稳定性的位点特异性单糖化蛋白。以溶菌酶和重组人成纤维细胞生长因子21(RhFGF21)为模型蛋白，我们发现IPPR同时完成了变性蛋白的聚乙二醇化和复性，复性产率和生物偶联效率都很高。IPPR聚乙二醇化的重组人成纤维细胞生长因子21具有与天然重组人成纤维细胞生长因子21相似的刺激3T3-L1细胞葡萄糖摄取的能力，但在肥胖/肥胖糖尿病小鼠模型中表现出延长的血糖和甘油三酯降低活性水平。因此，IPPR将极大地促进蛋白质疗法的产生。
Production of protein therapeutics often involves in vitro refolding from bacterial inclusion bodies and subsequent PEGylation to improve protein stability and plasma half-life. Here, we devised a novel strategy for one-step production of site-specific mono-PEGylated proteins with good bioactivity and improved biostability by integrating PEGylation and protein refolding (IPPR). Using lysozyme and recombinant human fibroblast growth factor 21 (rhFGF21) as model proteins, we showed that both PEGylation and refolding of denatured proteins have been simultaneously accomplished by IPPR with high efficiency of refolding yield and bioconjugation. PEGylated rhFGF21 by IPPR has a similar capacity as the native rhFGF21 to stimulate glucose uptake in 3T3-L1 cells, but exhibits prolonged blood glucose and triglyceride lowering activity levels in the ob/ob diabetic mouse model. Hence, IPPR will significantly facilitate the generation of protein therapeutics.