Centre for Vaccine Evaluation Biologics & Genetic Therapies Directorate, Health Canada, 251 Sir Frederick Banting Driveway, Ottawa, Ontario, K1A 0K9, Canada.
Pharm Res. 2011 Jul;28(7):1661-7. doi: 10.1007/s11095-011-0402-0. Epub 2011 Mar 9.
Thermal stability is considered an indication of protein fold and conformational stability. We investigate the influence of glycosylation on the thermal stability of interferon alpha 2b (IFN α-2b).
Far ultraviolet light circular dichroism spectroscopy (UV CD) and differential scanning calorimetry (DSC) were used to assess the thermal stability of the European Directorate for the Quality of Medicines IFN α-2b reference standards as well as an O-linked glycosylated IFN α-2b produced in human embryonic kidney cells.
Assessment of thermal stability of IFN α-2b and glycosylated IFN α-2b by DSC revealed that non-glycosylated interferon (Tm=65.7 +/- 0.2°C, n=3) was more thermally stable than the glycosylated variant (Tm=63.8 C +/- 0.4°C, n=3). These observations were confirmed with far UV CD (Tm IFN α-2b=65.3 +/- 0.4°C, Tm glycosylated IFN α-2b=63.6 +/- 0.2°C, n=3). Enzymatic deglycosylation of IFN α-2b resulted in improved thermally stability when assessed with far UV CD and DSC.
We demonstrate that O-linked glycosylation decreases the thermal stability of IFN α-2b compared to a non-glycosylated variant of the protein.
热稳定性被认为是蛋白质折叠和构象稳定性的指标。我们研究了糖基化对干扰素 alpha 2b（IFN α-2b）热稳定性的影响。
远紫外光圆二色性光谱（UV CD）和差示扫描量热法（DSC）用于评估欧洲药品质量局 IFN α-2b 参考标准以及在人胚肾细胞中产生的 O-连接糖基化 IFN α-2b 的热稳定性。
通过 DSC 评估 IFN α-2b 和糖基化 IFN α-2b 的热稳定性表明，非糖基化干扰素（Tm=65.7 +/- 0.2°C，n=3）比糖基化变体（Tm=63.8°C +/- 0.4°C，n=3）更热稳定。这些观察结果通过远紫外 CD 得到了证实（Tm IFN α-2b=65.3 +/- 0.4°C，Tm 糖基化 IFN α-2b=63.6 +/- 0.2°C，n=3）。用远紫外 CD 和 DSC 评估时，IFN α-2b 的酶促去糖基化导致热稳定性提高。
我们证明与蛋白质的非糖基化变体相比，O-连接糖基化降低了 IFN α-2b 的热稳定性。
