N-糖基化对TNFR-Fc融合蛋白结构稳定性和生物活性的影响

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。

The Impact of N-glycosylation on TNFR-Fc Fusion Protein Conformation Stability and Bioactivity

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.