The Use of Zwittergent 3-14 in the Purification of Recombinant Human Interferon-β Ser17(Betaseron)

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Abstract
A new method for purifying human interferon-β 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-β 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-β 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.