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中国生物工程杂志

CHINA BIOTECHNOLOGY
中国生物工程杂志
中国生物工程杂志  2016, Vol. 36 Issue (5): 12-19    DOI: 10.13523/j.cb.20160502
研究报告     
N-糖基化对TNFR-Fc融合蛋白结构稳定性和生物活性的影响
黄嘉慧, 汪才坤, 覃锦红, 陈龙冠, 黄云娜, 谢秋玲
暨南大学生命科学技术学院 广东省生物工程药物重点实验室 基因工程药物国家工程研究中心 广州 510632
The Impact of N-glycosylation on TNFR-Fc Fusion Protein Conformation Stability and Bioactivity
HUANG Jia-hui, WANG Cai-kun, QIN Jin-hong, CHEN Long-guan, HUANG Yun-na, XIE Qiu-ling
College of Life Science and Technology, Guangdong Provincial Key Laboratory of Bioengineering Medicine, National Engineering Research Center of Genetic Medicine, Jinan University, Guangzhou 510632, China
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摘要：

目的:探究糖基化对TNFR-Fc融合蛋白结构、稳定性和生物活性的影响。方法:经N-糖酰胺酶F(PNGase F)切除TNFR-Fc融合蛋白所连的糖链,用SEC-HPLC、傅里叶变换红外光谱法和荧光光谱法等方法分析N-糖基化和去糖基化后重组蛋白的结构变化,通过加速稳定性实验和毛细管电泳检测对比其酶切前后稳定性变化,其生物活性的差异经细胞杀伤实验进行比较。结果:去糖基化后TNFR-Fc融合蛋白质分子质量略有降低,其构象、荷电性质及生物活性没有明显差异;然而切除N-糖链,TNFR-Fc二聚体的稳定性降低,蛋白质降解物明显增加。结论:去N-糖基化对TNFR-Fc的构象、荷电性质和生物活性的影响并不显著,但会影响TNFR-Fc融合蛋白的稳定性。
关键词： 糖基化;  蛋白质构象;  去糖基化;  TNFR-Fc    
Abstract:

Object:To study the effect of glycosylation on the conformation, stability and bioactivity of recombinant TNFR-Fc fusion protein. Method:Using PNGase F to remove the oligosaccharide chains in the Fc region of TNFR-Fc fusion protein, and the differences between glycosylated and deglycosylated recombinant proteins were tested by size-exclusion chromatography(SEC), fourier transform infrared spectroscopy(FTIR)and fluorescent spectroscopy, followed by study of the thermal stability of the proteins. In addition, the difference of the bioacitivity by cell killing experiment is tested. Results:Although the molecular weight of TNFR-Fc became smaller after deglycosyltion, but there were no detected change of charge property, bioactivity, secondary or tertiary structural. However, deglycosylated TNFR-Fc exhibited less thermal stability. Conclusion:Deglycosylation didn't affect the conformation, bioactivity or charge property of TNFR-Fc, but induced a decrease in protein stability.
Key words: Protein conformation    Glycosylation    TNFR-Fc    Deglycosyltion
收稿日期: 2015-11-03 出版日期: 2016-01-04
ZTFLH:  Q591.2  
基金资助:

国家"十二五"重大新药创制项目(2012ZX09202-301-001),广东省战略性新兴产业核心技术攻关项目(2012A080800008),广东省重大科技专项(2012A080202014)资助项目
通讯作者: 谢秋玲     E-mail: txql@jnu.edu.cn
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黄嘉慧
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谢秋玲

引用本文:

黄嘉慧, 汪才坤, 覃锦红, 陈龙冠, 黄云娜, 谢秋玲. N-糖基化对TNFR-Fc融合蛋白结构稳定性和生物活性的影响[J]. 中国生物工程杂志, 2016, 36(5): 12-19.

HUANG Jia-hui, WANG Cai-kun, QIN Jin-hong, CHEN Long-guan, HUANG Yun-na, XIE Qiu-ling. The Impact of N-glycosylation on TNFR-Fc Fusion Protein Conformation Stability and Bioactivity. China Biotechnology, 2016, 36(5): 12-19.

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https://manu60.magtech.com.cn/biotech/CN/10.13523/j.cb.20160502    或    https://manu60.magtech.com.cn/biotech/CN/Y2016/V36/I5/12

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