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Purification and characterization of a long-acting ciliary neurotrophic factor via genetically fused with an albumin-binding domain
Xu, Longfu1,2; Zhang, Chun1; Liu, Liping1; Zhang, Yao1,2; Wang, Qi1,2; Wang, Jian3; Liu, Yongdong1; Su, Zhiguo1
2017-11-01
发表期刊PROTEIN EXPRESSION AND PURIFICATION
ISSN1046-5928
卷号139期号:NOV页码:14-20
摘要

Ciliary neurotrophic factor (CNTF) is a promising candidate for the treatment of neurodegenerative or metabolic diseases, but suffers rapid clearance in body. Herein we constructed a new long-acting recombinant human CNTF (rhCNTF) by genetic fusion with an albumin-binding domain (ABD) through a flexible peptide linker, hoping to endow the new molecule prolonged serum circulation time by binding with endogenous human serum albumin (HSA) and then utilizing the naturally long-half-life property of HSA. This fused protein rhCNTF-ABD was expressed in Escherichia coil mainly in the soluble form and purified through a two-step chromatography, with purity of 95% and a high yield of 90-100 mg/L culture. The in vitro binding ability of rhCNTF-ABD with HSA was firstly verified by incubation of the two components together followed by HP-SEC analysis. ABD-fused rhCNTF showed similar secondary and tertiary structure as the parent protein. It retained approximately 94.1% of the native bioactivity as demonstrated via CCK-8 cell viability assay analysis. In vivo studies in SD rats were performed and the terminal half-life of 483.89 min for rhCNTF-ABD was determined, which is about 14 folds longer than that of rhCNTF (34.28 min) and comparable with 20 k-40 kDa PEGylated rhCNTFs. The new constructed rhCNTF-ABD represents a potential therapeutic modality, and the proposed strategy may also have useful applications for other long-lasting biopharmaceutics' design. (C) 2017 Elsevier Inc. All rights reserved.

关键词Ciliary Neurotrophic Factor Albumin-binding Domain Pegylation Circulatory Half-life Pharmacokinetic
文章类型Article
WOS标题词Science & Technology ; Life Sciences & Biomedicine
DOI10.1016/j.pep.2017.07.006
收录类别SCI
语种英语
关键词[WOS]HALF-LIFE EXTENSION ; STREPTOCOCCAL PROTEIN-G ; HUMAN SERUM-ALBUMIN ; AFFIBODY MOLECULE ; NEURONOTROPHIC FACTOR ; ESCHERICHIA-COLI ; FUSION PROTEIN ; AFFINITY ; ANTIBODIES ; LIGANDS
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) ; Open Funding Project of the National Key Laboratory of Biochemical Engineering(2014KF-05)
WOS记录号WOS:000408400600003
引用统计
文献类型期刊论文
条目标识符http://ir.ipe.ac.cn/handle/122111/23203
专题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, 1 Beierjie St, Beijing 100190, Peoples R China
2.Univ Chinese Acad Sci, Beijing 100049, Peoples R China
3.Natl Vaccine & Serum Inst, Beijing 100024, Peoples R China
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GB/T 7714
Xu, Longfu,Zhang, Chun,Liu, Liping,et al. Purification and characterization of a long-acting ciliary neurotrophic factor via genetically fused with an albumin-binding domain[J]. PROTEIN EXPRESSION AND PURIFICATION,2017,139(NOV):14-20.
APA Xu, Longfu.,Zhang, Chun.,Liu, Liping.,Zhang, Yao.,Wang, Qi.,...&Su, Zhiguo.(2017).Purification and characterization of a long-acting ciliary neurotrophic factor via genetically fused with an albumin-binding domain.PROTEIN EXPRESSION AND PURIFICATION,139(NOV),14-20.
MLA Xu, Longfu,et al."Purification and characterization of a long-acting ciliary neurotrophic factor via genetically fused with an albumin-binding domain".PROTEIN EXPRESSION AND PURIFICATION 139.NOV(2017):14-20.
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