Identification of an oxidative refolding intermediate of recombinant consensus interferon from inclusion bodies and design of a two-stage strategy to promote correct disulfide-bond formation.
Dilution refolding of recombinant consensus IFN (interferon) from inclusion bodies suffers from low yield. A stable intermediate was found to mix with the correct product and to have an antiviral activity of less than 10% of the latter. This intermediate would form precipitates upon removal of the precipitation inhibitor arginine. Compared with the native protein, the intermediate moved more slowly on non-reducing SDS/PAGE. The CD and fluorescence spectra indicated that it had formed a native-like structure, but had only one disulfide bond: Cys(29)-Cys(139). Further evidence showed that the formation of Cys(29)-Cys(139) is specific and very likely to happen, even in the presence of a high concentration of reducing agent, whereas pairing of the other disulfide (Cys(1)-Cys(99)) needed a stronger oxidative condition. It competed with intermolecular disulfide bonding to form covalent oligomers. On the basis of this discovery, a two-stage refolding step strategy was designed that employed a modified dilution refolding step followed by a dialysis refolding step. The first stage used a high concentration of reducing agent together with the precipitation inhibitor arginine. The purpose was to hinder any reaction through Cys(1) or Cys(99) but allow the intramolecular disulfide bonding of Cys(29)-Cys(139). The second stage was a dialysis step that gradually increased the oxidative agent concentration and simultaneously decreased the arginine concentration. The refolding yield was increased from 35 to 82%, while the mass recovery was increased from 60 to 96%. Moreover, this strategy could suppress precipitation even after arginine was completely removed.
Liu
Yong-Dong
YD
National Key Laboratory of Biochemical Engineering, Institute of Process Engineering, Chinese Academy of Sciences, Beiertiao 1, Zhongguancun, Beijing 100080, People's Republic of China.
Zhang
Gui-Feng
GF
Li
Jing-Jing
JJ
Chen
Jing
J
Wang
Yin-Jue
YJ
Ding
Hong
H
Su
Zhi-Guo
ZG
D000595:Amino Acid Sequence; D001120:Arginine; D016384:Consensus Sequence; D004220:Disulfides / Q000737:chemistry*; D002479:Inclusion Bodies / Q000737:chemistry*; D007370:Interferon Type I / Q000737:chemistry* / Q000378:metabolism*; D010084:Oxidation-Reduction; D010446:Peptide Fragments / Q000737:chemistry; D011489:Protein Denaturation; D017510:Protein Folding; D020673:Protein Renaturation; D011994:Recombinant Proteins
D004220:Disulfides; D007370:Interferon Type I; D010446:Peptide Fragments; D011994:Recombinant Proteins; D001120:Arginine