Efficient preparation and PEGylation of recombinant human non-glycosylated erythropoietin expressed as inclusion body in E. coli
文献类型：期刊论文
| 作者 | Wang, Yin-Jue1,2; Liu, Yong-Dong1; Chen, Jing1,2; Hao, Su-Juan3; Hu, Tao1; Ma, Guang-Hui1; Su, Zhi-Guo1 |
| 刊名 | INTERNATIONAL JOURNAL OF PHARMACEUTICS
 |
| 出版日期 | 2010-02-15 |
| 卷号 | 386期号:1-2页码:156-164 |
| 关键词 | Recombinant erythropoietin Non-glycosylated Refolding Purification PEGylation Half-life |
| ISSN号 | 0378-5173 |
| 通讯作者 | Su, ZG |
| 英文摘要 | Recombinant human erythropoietin produced by mammalian cells contains about 40% carbohydrates which maintain its stability and long residence in body. However, mammalian derived Epo has low yields and high costs of production. In this article, a cost-effective strategy of producing non-glycosylated Epo from Escherichia coli and then PEGylating it to replace the role of sugar chains was investigated. Recombinant human non-glycosylated erythropoietin (rh-ngEpo) was overexpressed as inclusion body in E. coli. As the routine inclusion body washing step resulted in poor protein recovery and purity, a new process scheme of using strong ion-exchange chromatography to purify denatured rh-ngEpo from inclusion body before refolding was developed. The purity of the denatured rh-ngEpo was increased from 59% to over 90%. Rh-ngEpo was then refolded and subsequently purified by one step of weak cation-exchange chromatography to 98% pure. Final protein yield was 129 mg/l, a significant improvement from 49 mg/l obtained via the conventional practice. The in vitro bioactivity of purified rh-ngEpo was comparable with the CHO-expressed Epo and the formation of native secondary structure was also confirmed by CD spectra. Rh-ngEpo was then modified by a 20 kDa methoxy polyethylene glycol (PEG) succinimidyl carbonate. The monoPEGylated protein, which retained 68% bioactivity, had enhanced thermal stability and a remarkably prolonged circulating half-life in rats as compared with that of the unmodified protein. These studies demonstrated the feasibility of PEGylating rh-ngEpo as a promising way for the development of new Epo drugs. (C) 2009 Elsevier B.V. All rights reserved. |
| WOS标题词 | Science & Technology ; Life Sciences & Biomedicine |
| 类目[WOS] | Pharmacology & Pharmacy |
| 研究领域[WOS] | Pharmacology & Pharmacy |
| 关键词[WOS] | CELL-LINE ; CHO-CELLS ; AGGREGATION ; CARBOHYDRATE ; PURIFICATION ; PROTEINS ; BODIES ; SITES |
| 收录类别 | SCI |
| 语种 | 英语 |
| WOS记录号 | WOS:000274876400019 |
| 公开日期 | 2013-11-27 |
| 版本 | 出版稿 |
| 源URL | [http://ir.ipe.ac.cn/handle/122111/6211]  |
| 专题 | 过程工程研究所_生化工程国家重点实验室 |
| 作者单位 | 1.Chinese Acad Sci, Inst Proc Engn, Natl Key Lab Biochem Engn, Beijing 100190, Peoples R China 2.Chinese Acad Sci, Grad Sch, Beijing 100190, Peoples R China 3.Beijing Univ Chem Technol, Beijing 100029, Peoples R China |
| 推荐引用方式 GB/T 7714 | Wang, Yin-Jue,Liu, Yong-Dong,Chen, Jing,et al. Efficient preparation and PEGylation of recombinant human non-glycosylated erythropoietin expressed as inclusion body in E. coli[J]. INTERNATIONAL JOURNAL OF PHARMACEUTICS,2010,386(1-2):156-164. |
| APA | Wang, Yin-Jue.,Liu, Yong-Dong.,Chen, Jing.,Hao, Su-Juan.,Hu, Tao.,...&Su, Zhi-Guo.(2010).Efficient preparation and PEGylation of recombinant human non-glycosylated erythropoietin expressed as inclusion body in E. coli.INTERNATIONAL JOURNAL OF PHARMACEUTICS,386(1-2),156-164. |
| MLA | Wang, Yin-Jue,et al."Efficient preparation and PEGylation of recombinant human non-glycosylated erythropoietin expressed as inclusion body in E. coli".INTERNATIONAL JOURNAL OF PHARMACEUTICS 386.1-2(2010):156-164. |
入库方式： OAI收割
来源：过程工程研究所
浏览0
下载0
收藏0
其他版本
除非特别说明，本系统中所有内容都受版权保护，并保留所有权利。