High level soluble expression, purification, and characterization of human ciliary neuronotrophic factor in Escherichia coli by single protein production system.

Abstract

Ciliary neurotrophic factor (CNTF) is characterized as a neuropoietic cytokine for a broad spectrum of neurons, leading to its evaluation in humans suffering from neurodegenerative diseases. Due to its wide range of biological applications, high yield production of soluble biologically active recombinant human CNTF (rhCNTF) in heterologous expression system is demanded. Many attempts had been undertaken to product rhCNTF in Escherichia coli (E. coli), however, the expression level of rhCNTF was low and most of which formed insoluble inclusion bodies. In this study, we described a new and efficient method to express rhCNTF. The human CNTF gene was codon optimized and then expressed by the single protein production (SPP) expression system in E. coli. The results showed that rhCNTF was expressed as a soluble biologically active protein, and upon purification, the final yield was about 250 mg/L in shake flask with a specific neuroprotective activity in Aβ-induced SH-SY5Y cell injury model. Our study might open up a new strategy for large-scale production of functional rhCNTF for clinical applications as well as basic research.

• Authors: Ke Wang, Fanfan Zhou, Lan Zhu, Xue Zhu, Kai Zhang, Ling Zhu
• Journal: Protein expression and purification (Protein Expr Purif) Vol. 96 Pg. 8-13 (Apr 2014) ISSN: 1096-0279 [Electronic] United States
• PMID: 24486790 (Publication Type: Journal Article, Research Support, Non-U.S. Gov't)
• Copyright: Copyright © 2014 Elsevier Inc. All rights reserved.

Chemical References

• Ciliary Neurotrophic Factor

Topics

• Base Sequence
• Ciliary Neurotrophic Factor (biosynthesis, genetics)
• Cloning, Molecular
• Cytoprotection
• Escherichia coli (genetics, metabolism)
• Gene Expression
• Genetic Therapy
• Humans
• Molecular Sequence Data
• Neurodegenerative Diseases (therapy)
• Plasmids (genetics)