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High hydrostatic pressure enables almost 100% refolding of recombinant human ciliary neurotrophic factor from inclusion bodies at high concentration
Wang, Qi1,2; Liu, Yongdong1; Zhang, Chun1; Guo, Fangxia1,2; Feng, Cui1; Li, Xiunan1; Shi, Hong1; Su, Zhiguo1
2017-05-01
发表期刊PROTEIN EXPRESSION AND PURIFICATION
ISSN1046-5928
卷号133页码:152-159
摘要Protein refolding from inclusion bodies (IBs) often encounters a problem of low recovery at high protein concentration. In this study, we demonstrated that high hydrostatic pressure (HHP) could simultaneously achieve high refolding concentration and high refolding yield for IBs of recombinant human ciliary neurotrophic factor (rhCNTF), a potential therapeutic for neurodegenerative diseases. The use of dilution refolding obtained 18% recovery at 3 mg/mL, even in the presence of 4 M urea. In contrast, HHP refolding could efficiently increase the recovery up to almost 100% even at 4 mg/mL. It was found that in the dilution, hydrophobic aggregates were the off-path products and their amount increased with the protein concentration. However, HHP could effectively minimize the formation of hydrophobic aggregates, leading to almost complete conversion of the rhCNTF IBs to the correct configuration. The stable operation range of concentration is 0.5-4.0 mg/mL, in which the refolding yield was almost 100%. Compared with the literatures where HHP failed to increase the refolding yield beyond 90%, the reason could be attributed to the structural difference that rhCNTF has no disulfide bond and is a monomeric protein. After purification by one-step of anionic chromatography, the purity of rhCNTF reached 95% with total process recovery of 54.1%. The purified rhCNTF showed similar structure and in vitro bioactivity to the native species. The whole process featured integration of solubilization/refolding, a high refolding yield of 100%, a high concentration of 4 mg/mL, and a simple chromatography to ensure a high productivity. (C) 2017 Elsevier Inc. All rights reserved.
关键词Rhcntf High Hydrostatic Pressure Inclusion Bodies Hydrophobic Aggregation Refolding
文章类型Article
WOS标题词Science & Technology ; Life Sciences & Biomedicine
DOI10.1016/j.pep.2017.03.014
收录类别SCI
语种英语
关键词[WOS]FACTOR CNTF ; ESCHERICHIA-COLI ; GROWTH-HORMONE ; PROTEINS ; PURIFICATION ; EXPRESSION ; COMPRESSIBILITY ; SOLUBILIZATION ; OPTIMIZATION ; WEIGHT
WOS研究方向Biochemistry & Molecular Biology ; Biotechnology & Applied Microbiology
WOS类目Biochemical Research Methods ; Biochemistry & Molecular Biology ; Biotechnology & Applied Microbiology
项目资助者National Natural Science Foundation of China(21576267 ; Beijing Natural Science Foundation(2162041) ; Major State Basic Research Development Program of China(2013CB733604) ; National Key Laboratory of Biochemical Engineering(2014KF-05) ; 21506228)
WOS记录号WOS:000401684400019
引用统计
文献类型期刊论文
条目标识符http://ir.ipe.ac.cn/handle/122111/22544
专题生化工程国家重点实验室
作者单位1.Chinese Acad Sci, Natl Key Lab Biochem Engn, Inst Proc Engn, 1 Beierjie St, Beijing 100190, Peoples R China
2.Univ Chinese Acad Sci, Beijing 100049, Peoples R China
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Wang, Qi,Liu, Yongdong,Zhang, Chun,et al. High hydrostatic pressure enables almost 100% refolding of recombinant human ciliary neurotrophic factor from inclusion bodies at high concentration[J]. PROTEIN EXPRESSION AND PURIFICATION,2017,133:152-159.
APA Wang, Qi.,Liu, Yongdong.,Zhang, Chun.,Guo, Fangxia.,Feng, Cui.,...&Su, Zhiguo.(2017).High hydrostatic pressure enables almost 100% refolding of recombinant human ciliary neurotrophic factor from inclusion bodies at high concentration.PROTEIN EXPRESSION AND PURIFICATION,133,152-159.
MLA Wang, Qi,et al."High hydrostatic pressure enables almost 100% refolding of recombinant human ciliary neurotrophic factor from inclusion bodies at high concentration".PROTEIN EXPRESSION AND PURIFICATION 133(2017):152-159.
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