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Purification and characterization of a long-acting ciliary neurotrophic factor via genetically fused with an albumin-binding domain.

Abstract：

: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 coli 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.

journal_name

Protein Expr Purif

journal_title

Protein expression and purification

authors

Xu L,Zhang C,Liu L,Zhang Y,Wang Q,Wang J,Liu Y,Su Z

doi

10.1016/j.pep.2017.07.006

subject

Has Abstract

pub_date

2017-11-01 00:00:00

pages

14-20

eissn

1046-5928

issn

1096-0279

pii

S1046-5928(17)30158-4

journal_volume

139

pub_type

杂志文章

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