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Efficient preparation and PEGylation of recombinant human non-glycosylated erythropoietin expressed as inclusion body in E. coli
Wang, Yin-Jue1,2; Liu, Yong-Dong1; Chen, Jing1,2; Hao, Su-Juan3; Hu, Tao1; Ma, Guang-Hui1; Su, Zhi-Guo1; Su, ZG
2010-02-15
Source PublicationINTERNATIONAL JOURNAL OF PHARMACEUTICS
ISSN0378-5173
Volume386Issue:1-2Pages:156-164
AbstractRecombinant 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.
KeywordRecombinant Erythropoietin Non-glycosylated Refolding Purification Pegylation Half-life
SubtypeArticle
WOS HeadingsScience & Technology ; Life Sciences & Biomedicine
DOI10.1016/j.ijpharm.2009.11.016
Indexed BySCI
Language英语
WOS KeywordCELL-LINE ; CHO-CELLS ; AGGREGATION ; CARBOHYDRATE ; PURIFICATION ; PROTEINS ; BODIES ; SITES
WOS Research AreaPharmacology & Pharmacy
WOS SubjectPharmacology & Pharmacy
WOS IDWOS:000274876400019
Citation statistics
Cited Times:25[WOS]   [WOS Record]     [Related Records in WOS]
Document Type期刊论文
Version出版稿
Identifierhttp://ir.ipe.ac.cn/handle/122111/6211
Collection生化工程国家重点实验室
Corresponding AuthorSu, ZG
Affiliation1.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
Recommended Citation
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, ZG.(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.
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