Russell-Harde D, Knauf M, Croze E
Department of Protein Biochemistry and Biophysics, Berlex Biosciences, Inc., Richmond, CA 94804-0099, USA.
J Interferon Cytokine Res. 1995 Jan;15(1):31-7. doi: 10.1089/jir.1995.15.31.
A new method for purifying human interferon-beta SER17 from E. coli-derived inclusion bodies has been developed. This procedure eliminates the need for strong denaturants, such as sodium dodecyl sulfate or chaotropes. The procedure makes use of a nondenaturing detergent and a brief incubation at pH 12 to solubilize interferon-beta Ser17 from inclusion bodies. The detergent used was Zwittergent 3-14 (nonionic and pH-insensitive), which is included in the class of sulfobetaines (RN+ (CH3)2(CH2)xSO3-). Zwittergent 3-14 was used in combination with urea to produce a urea/Zwittergent 3-14 washed inclusion body preparation enriched in human interferon-beta Ser17 (Betaseron). Solubilization of inclusion bodies was accomplished by employing a brief (1 minute) shift to pH 12 in the presence of 2.5% Zwittergent 3-14 followed by rapid adjustment to pH 8.0. Solubilization was complete, and the solution could be rapidly adjusted to pH 8 without any observable precipitation of protein. The resultant supernatant could be successfully subjected to a number of chromatographic and analytic procedures, many of which are not compatible with strong anionic detergents, such as SDS. Betaseron was purified from Zwittergent 3-14 solubilized inclusion body lysates using both ion-exchange and size-exclusion chromatography. Purified Betaseron retained bioactivity and could be refolded by simple dialysis against a nonreducing buffer. This method represents a novel procedure for purifying Betaseron from inclusion bodies using a nondenaturing detergent and ion-exchange chromatography.
已开发出一种从大肠杆菌来源的包涵体中纯化人β-干扰素SER17的新方法。该程序无需使用强变性剂，如十二烷基硫酸钠或离液剂。该程序利用非变性去污剂并在pH 12下短暂孵育，以使β-干扰素Ser17从包涵体中溶解。所使用的去污剂是两性离子去污剂3-14（非离子且对pH不敏感），属于磺基甜菜碱类（RN+(CH3)2(CH2)xSO3-）。两性离子去污剂3-14与尿素联合使用，以制备富含人β-干扰素SER17（倍泰龙）的尿素/两性离子去污剂3-14洗涤包涵体制剂。通过在2.5%两性离子去污剂3-14存在下短暂（1分钟）将pH值调至12，然后迅速调至pH 8.0，实现包涵体的溶解。溶解完成后，溶液可迅速调至pH 8，而不会观察到蛋白质沉淀。所得上清液可成功进行多种色谱和分析程序，其中许多程序与强阴离子去污剂（如SDS）不兼容。使用离子交换色谱和尺寸排阻色谱从两性离子去污剂3-14溶解的包涵体裂解物中纯化倍泰龙。纯化的倍泰龙保留了生物活性，并且可以通过在非还原缓冲液中简单透析进行复性。该方法代表了一种使用非变性去污剂和离子交换色谱从包涵体中纯化倍泰龙的新程序。
