The use of Zwittergent 3-14 in the purification of recombinant human interferon-beta Ser17 (Betaseron).
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.
Russell-Harde
D
D
Department of Protein Biochemistry and Biophysics, Berlex Biosciences, Inc., Richmond, CA 94804-0099, USA.
Knauf
M
M
Croze
E
E
D003902:Detergents; D004926:Escherichia coli; D006801:Humans; D002479:Inclusion Bodies / Q000737:chemistry*; D000068556:Interferon beta-1a; D000068576:Interferon beta-1b; D016899:Interferon-beta / Q000302:isolation & purification*; D017510:Protein Folding; D000644:Quaternary Ammonium Compounds / Q000494:pharmacology*; D011994:Recombinant Proteins / Q000302:isolation & purification; D012995:Solubility
D003902:Detergents; D000644:Quaternary Ammonium Compounds; D011994:Recombinant Proteins; D000068576:Interferon beta-1b; C048256:zwittergent 3-14; D016899:Interferon-beta; D000068556:Interferon beta-1a