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
文献类型：期刊论文
| 作者 | Liu, Yong-Dong; Zhang, Gui-Feng; Li, Jing-Jing; Chen, Jing; Wang, Yin-Jue; Ding, Hong; Su, Zhi-Guo |
| 刊名 | BIOTECHNOLOGY AND APPLIED BIOCHEMISTRY
 |
| 出版日期 | 2007-12-01 |
| 卷号 | 48期号:s页码:189-198 |
| 关键词 | performance liquid-chromatography pancreatic trypsin-inhibitor human-leukocyte interferons human alpha-interferon egg-white lysozyme escherichia-coli structural characterization purification proteins renaturation |
| ISSN号 | 0885-4513 |
| 其他题名 | Biotechnol. Appl. Biochem. |
| 中文摘要 | 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 3S to 82%, while the mass recovery was increased from 60 to 96%. Moreover, this strategy could suppress precipitation even after arginine was completely removed. |
| 英文摘要 | 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 3S to 82%, while the mass recovery was increased from 60 to 96%. Moreover, this strategy could suppress precipitation even after arginine was completely removed. |
| WOS标题词 | Science & Technology ; Life Sciences & Biomedicine |
| 类目[WOS] | Biochemistry & Molecular Biology ; Biotechnology & Applied Microbiology |
| 研究领域[WOS] | Biochemistry & Molecular Biology ; Biotechnology & Applied Microbiology |
| 关键词[WOS] | PERFORMANCE LIQUID-CHROMATOGRAPHY ; PANCREATIC TRYPSIN-INHIBITOR ; HUMAN-LEUKOCYTE INTERFERONS ; HUMAN ALPHA-INTERFERON ; EGG-WHITE LYSOZYME ; ESCHERICHIA-COLI ; STRUCTURAL CHARACTERIZATION ; PURIFICATION ; PROTEINS ; RENATURATION |
| 收录类别 | SCI |
| 原文出处 | |
| 语种 | 英语 |
| WOS记录号 | WOS:000251883000008 |
| 公开日期 | 2013-10-15 |
| 版本 | 出版稿 |
| 源URL | [http://ir.ipe.ac.cn/handle/122111/3398]  |
| 专题 | 过程工程研究所_研究所（批量导入） |
| 作者单位 | Chinese Acad Sci, Inst Proc Engn, Natl Key Lab Biochem Engn, Beijing 100080, Peoples R China |
| 推荐引用方式 GB/T 7714 | Liu, Yong-Dong,Zhang, Gui-Feng,Li, Jing-Jing,et al. 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[J]. BIOTECHNOLOGY AND APPLIED BIOCHEMISTRY,2007,48(s):189-198. |
| APA | Liu, Yong-Dong.,Zhang, Gui-Feng.,Li, Jing-Jing.,Chen, Jing.,Wang, Yin-Jue.,...&Su, Zhi-Guo.(2007).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.BIOTECHNOLOGY AND APPLIED BIOCHEMISTRY,48(s),189-198. |
| MLA | Liu, Yong-Dong,et al."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".BIOTECHNOLOGY AND APPLIED BIOCHEMISTRY 48.s(2007):189-198. |
入库方式： OAI收割
来源：过程工程研究所
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